Pro-inflammatory and anti-cancer functions of toll-like receptor 4 antagonists

ABSTRACT

The present invention provides methods and compositions for specific activation of inflammatory responses in dendritic cells (DCs). 1-palmitoyl-2-arachidonyl-sn-glycero phosphorylcholine (PAPC) and its oxidized variant (oxPAPC) were identified to promote DC-mediated immunity, and are provided as adjuvants in immunostimulatory compositions, including vaccines.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/730,030, filed on Apr. 26, 2022, which is a continuation of U.S.patent application Ser. No. 17/018,045, filed Sep. 11, 2020 and U.S.patent application Ser. No. 17/018,038, filed on Sep. 11, 2020, whichare continuations of U.S. patent application Ser. No. 15/543,165, filedon Jul. 12, 2017, which is a National Phase application filed under 35U.S.C. § 371 of International Patent Application No. PCT/US2016/012994with an International Filing Date of Jan. 12, 2016, which claims thebenefit of priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 62/102,245, filed Jan. 12, 2015 and entitled,“Pro-Inflammatory and Adjuvant Functions of Toll-Like Receptor 4Antagonists.” The entire content of the foregoing are incorporatedherein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under grant numberAI103082-01A1, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the fields of adjuvants,immunostimulation and vaccines.

BACKGROUND OF THE INVENTION

The ability to distinguish self from non-self molecules is a fundamentalfeature of all forms of life, yet our understanding of this distinctionremains incomplete. In mammals, it is commonly believed that the PatternRecognition Receptors (PRRs) of the innate immune system serve thefunction of distinguishing self and non-self molecules. This idea, firstput forth by Charles Janeway Jr., has been extensively validated throughthe study of various families of PRRs, such as the Toll-like Receptors(TLRs), RIG-I like Receptors (RLRs), NOD-like Receptors (NLR) and C-typeLectin Receptors (CLRs) (Iwasaki, A., and Medzhitov, R. (2015) NatImmunol 16, 343-353). PRRs act to either directly or indirectly detectmolecules that are common to broad classes of microbes. These moleculesare classically referred to as pathogen associated molecular patterns(PAMPs), and include factors such as bacterial lipopolysaccharides(LPS), bacterial flagellin or viral double stranded RNA, among others(Janeway, C. A., Jr. (1989) Spring Harb Symp Quant Biol 54 Pt 1, 1-13).Detection of microbial products activates PRR-dependent cellularresponses that are either pro-inflammatory or immuno-regulatory, withthe best example of the latter being the activation of antigen-specificT-cells to promote adaptive immunity (Iwasaki, A., and Medzhitov, R.(2015) Nat Immunol 16, 343-353). PRR-mediated pro-inflammatory responsescan be considered those that occur in numerous types of cells, whereasactivities designed to promote T-cell activation often occur uniquely indendritic cells (DCs). DC-specific activities induced by PRRs includethe acidification of endosomes and phagosomes (Delamarre, L. et al.,(2005) Science 307, 16301634, and Trombetta, E. S. et al., (2003)Science 299, 1400-1403), delivery of major histocompatibility complex(MHC) molecules to microbe-containing phagosomes (Nair-Gupta, P. et al.(2014) Cell 158, 506521), loading of microbial peptides on MHC, anddelivery of MHC molecules to the cell surface (Blander, J. M., andMedzhitov, R. (2006). Nature 440, 808-812; Inaba, K. et al., (2000) JExp Med 191, 927-936; Pierre, P. et al., (1997) Nature 388, 787-792;Turley, S. J. et al., (2000) Science 288, 522-527). All of theseactivities promote effective antigen presentation to T-cells and theinitiation of adaptive immunity.

Adjuvants are substances that accelerate and/or enhance an antigenspecific immune response. The purpose of an adjuvant is to make anantigen visible to the eyes (macrophages/dendritic cells) of the immunesystem. Recognition of antigens by antigen presenting cells (APCs) suchas macrophages and dendritic cells essentially initiates the criticalcascade of events leading to localized inflammation, which recruits APCsand ultimately leads to initiation of a productive cell mediated and/orantibody mediated immune response. Currently, the majority of humanvaccines contain aluminum salts as an adjuvant and pharmaceuticalcompanies are developing oil-based adjuvants to be incorporated intovaccines. For development of improved immunostimulatory compositions(e.g., vaccines), identification and inclusion of adjuvants thatselectively activate dendritic cells (DC) while minimally activatingmacrophages would be beneficial in reducing adverse side effects ofadministering such compositions, such as malaise and inflammation.Currently, adjuvants which act as agonists to TLR-2, TLR-5, TLR7/8, andTLR-9 are being studied, and one TLR-4 agonist, monophosphoryl lipid A,is FDA approved.

SUMMARY OF THE INVENTION

The invention is based, at least in part, upon the discovery thatendogenous oxidized phospholipids, which are Toll-like Receptor (TLR)antagonists that are found at sites of tissue damage, created adendritic cell state that was hyper-inflammatory. It was specificallydemonstrated that, in a context-dependent manner, oxPAPC induced severalresponses within DCs that promoted their ability to activate antigenspecific T-cells. These findings indicated that phospholipids such asoxPAPC (and related phospholipids capable of activating non-canonicalinflammasomes, as well as, e.g., Rhodo LPS, which was also found toactivate non-canonical inflammasomes) could function as an enhancedclass of adjuvants for use in prophylactic and therapeuticimmunostimulatory compositions.

In the presence of diverse TLR ligands, oxPAPC was identified to promoteDC survival and trigger the release of the T-cell activating cytokineinterleukin 1 beta (IL-1β). Mechanistically, oxPAPC was characterized asengaging the LPS receptor CD14 on the surface of DCs, which allowed itsdelivery into endosomes and subsequent access to the cytosolic proteincaspase-11. Caspase-11 engagement by oxPAPC triggered theinflammasome-mediated release of IL-1β. These oxPAPC-triggered responsesdid not occur in macrophages, indicating that the actions of this lipidwere uniquely designed to promote immuno-regulatory activities of DCs,as opposed to general (macrophage-mediated) inflammatory responses.Consequently, oxPAPC was identified to synergize with microbial productsto induce a more robust activation of antigen specific T-cells thancould be elicited by PAMPs alone. These molecules, (dubbed vita-DAMPs),were identified as functioning together with PAMPs to hyperactivate DCsand elicit maximal adaptive immune responses.

In one aspect, the invention provides a composition for eliciting animmune response to an immunogen that includes an immunogen and anon-canonical inflammasome-activating lipid.

In one embodiment, the non-canonical inflammasome-activating lipid isoxPAPC. In another embodiment, the non-canonical inflammasome-activatinglipid is PAPC. Optionally, the non-canonical inflammasome-activatinglipid is one or more species of oxPAPC. In a related embodiment, thenon-canonical inflammasome-activating lipid is one or more of HOdiA-PC,KOdiA-PC, HOOA-PC and KOOA-PC. In another embodiment, the non-canonicalinflammasome-activating lipid is Rhodo LPS.

In an additional embodiment, the non-canonical inflammasome-activatinglipid enhances an immune response to the immunogen when the compositionis administered to a subject, as compared to a composition lacking thenon-canonical inflammasome-activating lipid.

In one embodiment, the immunogen and lipid are present at aconcentration sufficient to induce dendritic cell (DC) activation whenthe composition is administered to a subject.

Optionally, the composition does not elicit a macrophage inflammatoryresponse when administered to a subject.

In one embodiment, the immunogen includes a human papilloma virusantigen, a herpes virus antigen such as herpes simplex antigen or herpeszoster antigen, a retrovirus antigen such as human immunodeficiencyvirus 1 antigen or human immunodeficiency virus 2 antigen, a hepatitisvirus antigen, an influenza virus antigen, a rhinovirus antigen,respiratory syncytial virus antigen, cytomegalovirus antigen, adenovirusantigen, Mycoplasma pneumoniae antigen, an antigen of a bacterium of thegenus Salmonella, Staphylococcus, Streptococcus, Enterococcus,Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, an amoebaantigen, a malarial parasite antigen and/or a Trypanosoma cruzi antigen.

Optionally, the composition is lyophilized.

In another embodiment, the composition consists essentially of theimmunogen in combination with the non-canonical inflammasome-activatinglipid.

Another aspect of the invention provides a pharmaceutical compositionthat includes an immunogen-adjuvant composition of the invention and apharmaceutically acceptable carrier.

In one embodiment, the carrier is an aqueous carrier. In anotherembodiment, the carrier is a solid carrier.

A further aspect of the invention provides a method for inducing aninflammatory response in a dendritic cell of a subject that includesadministering the composition of claim 1 to the subject.

An additional aspect of the invention provides a method for enhancing aprotective immune response to an immunogen in a subject, byadministering the immunogen and a non-canonical inflammasome-activatinglipid to a subject in an amount effective to enhance a protective immuneresponse in the subject, where the non-canonical inflammasome-activatinglipid is administered in an adjuvant-effective amount.

In one embodiment, the immunogen and the non-canonicalinflammasome-activating lipid are administered concurrently to thesubject.

Another aspect of the invention provides a method of inducing an immuneresponse in a subject that includes concurrently administering animmunogen and a non-canonical inflammasome-activating lipid to thesubject in an amount effective to produce an immune response in thesubject.

In one embodiment, the subject is human.

In another embodiment, the immunogen and the non-canonicalinflammasome-activating lipid are administered simultaneously in acommon pharmaceutical carrier.

Optionally, the immunogen and the non-canonical inflammasome-activatinglipid are administered by parenteral administration.

In one embodiment, the immune response is a prophylactic immuneresponse.

In another embodiment, the immune response is a therapeutic immuneresponse.

In an additional embodiment, the immune response includes a humoralimmune response.

Other aspects of the invention are described in, or are obvious from,the following disclosure, and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that PAPCs (e.g., oxPAPC) were identified to act asTLR4-antagonists and not as TLR4-agonists.

FIG. 2 shows that different doses of PAPCs modulated TLR4 signaling.

FIG. 3 shows that different doses of PAPCs modulated CD14 levels on theplasma membrane.

FIG. 4 shows that different doses of PAPCs modulated LPS-dependent TLR4internalization.

FIG. 5 shows that different doses of PAPCs modulated LPS-dependent TLR4dimerization.

FIG. 6 shows that PAPCs activated the inflammasome in DCs.

FIG. 7 shows that KOdiA-PC activated the inflammasome.

FIG. 8 shows that CD14 regulated inflammasome activation in response toPAPCs.

FIG. 9 shows that inflammasome activation in response to PAPCs was CD14specific, although PAPCs could also induce CD36 internalization.

FIG. 10 shows that CD14 regulated PAPCs-mediated inflammasome activationindependently of Type I IFNs.

FIG. 11 shows that regulation of Caspase-1 and Caspase-11 expression wassimilar in wtDCs and Cd14−/−DCs.

FIG. 12 shows that PAPCs induced inflammasome activation in a cell typespecific manner.

FIG. 13 shows that other PAMPs primed PAPCs-induced inflammasomeactivation.

FIG. 14 shows additional results that other PAMPs primed PAPCs-inducedinflammasome activation.

FIG. 15 shows that not all modified PCs induced inflammasome activation.

FIG. 16 shows that Nlrp3 was required to induce inflammasome activation.

FIG. 17 shows that Asc was required to induce inflammasome activation.

FIG. 18 shows that Casp1/Casp11 were required to induce inflammasomeactivation.

FIG. 19 shows that oxPAPC-induced inflammasome activation, but notATP-induced inflammasome activation, was Caspase-11 dependent.

FIG. 20 shows that PAPCs-induced inflammasome activation after primingwith both LPS and Pam3 required Caspase-11.

FIG. 21 shows that biotinylated PAPCs potently induced CD14internalization.

FIG. 22 shows that biotinylated PAPCs did not induce TLR4internalization.

FIG. 23 shows that biotinylated PAPCs did not induce IL-1b secretion.

FIG. 24 shows in vitro binding assays for biotinylated LPS, OxPac andPac to Caspase 11 and MD-2, which appeared to identify complex formationin a caspase 11-dependent manner.

FIG. 25 shows that in a Bio-LPS pull-down, PAPC acted as adose-dependent competitor. Biotin-LPS was used at 5 μg per pull-downassay. PAPC was used at 5, 50 and 500 μg to compete with LPS binding toMD-2 and Caspase-11, respectively. The competition started to beeffective at the ratio of 1:100 (LPS:PAPC).

FIG. 26 shows that oxPAPC and LPS likely bound the same domain of CD14.

FIG. 27 shows that LPS treatment affected DC survival.

FIG. 28 shows that PAPCs treatment of primed DCs favored DC survival.

FIG. 29 shows that the observed PAPCs-dependent pro-survival effect wasnot CD14-dependent.

FIG. 30 shows that P2C and P3C alone supported DC survival.

FIG. 31 shows that P2C and P3C primed DC did not increase their survivalin response to PAPCs treatment.

FIG. 32 shows that the inflammasome was efficiently activated byco-administration of priming stimuli and PAPCs but not ATP.

FIG. 33 shows that co-administration of priming stimuli and PAPCs didnot alter NF-κB activation in wtDC.

FIG. 34 shows that oxPAPC acted as an antagonist of TLR4 signaling inthe absence of CD14.

FIG. 35 shows that co-administration of LPS and oxPAPC affected TLR4internalization.

FIG. 36 shows that co-administration of LPS and oxPAPC affected CD14internalization.

FIG. 37 shows that co-administration of LPS and oxPAPC partiallyaffected TLR4 dimerization.

FIG. 38 shows that Rhodo LPS was a potent inducer of inflammasomeactivation.

FIG. 39 shows that LPS-induced inflammasome activation wasNlrp3-dependent.

FIG. 40 shows that LPS-induced inflammasome activation wasAsc-dependent.

FIG. 41 shows that LPS-induced inflammasome activation wasCasp1/11-dependent.

FIG. 42 shows that Rhodo LPS-induced inflammasome activation wasCD14-independent.

FIGS. 43A-43D depict images showing that oxPAPC did not bind TLR4 orinduce TL4F signaling. FIG. 43A presents a line graph showing the extentof TLR4 dimerization in iMΦs treated with LPS (1 μg/ml) or oxPAPC (50μM) for the indicated time points. TLR4 dimerization was measured byflow cytometry. The line graph represents means and standard deviationsof two independent experiments. FIG. 43B depicts a line graph showingIL-1β, IL-6, IFNβ and Viperin levels in iMΦs treated with LPS (1 μg/ml)or oxPAPC (50 μM). Gene expression relative to GAPDH was analyzed byqPCR at times indicated. Untreated cells were used as a negative controlin all experiments. Line graphs represent the average and error barsrepresent the standard deviation of triplicate readings from onerepresentative experiment of three. FIG. 43C depicts a blot showingmyddosome formation in at the indicated time points after treatment withLPS (1 μg/ml) or oxPAPC (50 μM) by co-immunoprecipitation (IP) of IRAK4with MyD88 followed by Western analysis of the proteins indicated. FIG.43D depicts a blot showing whole cell lysates (WCL) collected and DCsmonitored for STAT-1 phosphorylation and viperin expression aftertreatment with LPS (1 μg/ml) or oxPAPC (50 μM).

FIGS. 44A-44F depict images showing that oxPAPC acted as both a CD14agonist and a TLR4 antagonist. FIG. 44A depicts a line graph indicatingsurface levels of CD14 and TLR4 in iMΦ lines treated with LPS (1 μg/ml)or oxPAPC (50 μM) for the indicated times. Surface levels of CD14 andTLR4 were measured by flow cytometry. Line graphs represent means andstandard deviations of two independent experiments. FIG. 44B depicts aline graph indicating results for primary DCs and MΦs treated withoxPAPC (50 μM) for the indicated times. Surface levels of CD14 and TLR4and TLR4 dimerization were measured by flow cytometry. Line graphsrepresent means and standard deviations of two independent experiments.FIG. 44C depicts a line graph indicating results for treated with LPSalone (1 μg/ml), oxPAPC alone (at the indicated concentration) orpre-treated with oxPAPC for 30′ and then with LPS. Surface levels ofTLR4 and TLR4 dimerization were measured by flow cytometry. Line graphsrepresent means and standard deviations of biological duplicates fromone experiment representative of three. FIG. 44D depicts images showingresults for treated with LPS alone (at the indicated concentrations),oxPAPC alone (120 μM) or pre-treated with oxPAPC for 30′ and then withLPS. FIG. 44D (left panel) shows TNFα secretion measured 18 hours afterLPS stimulation by ELISA. Line graphs represent the average and errorbars represent the standard deviation of triplicate readings from onerepresentative experiment of three. FIG. 44D (right panels) shows STAT-1phosphorylation measured 4 hours after LPS treatment by Westernanalysis. FIG. 44E depicts a blot showing the oxPAPC binding capacity ofthe CD14 mutants was determined by biotinylated oxPAPC pull down assay.Lysates of 293T cells expressing the indicated CD14 mutants wereincubated with biotinylated oxPAPC (10 μg). CD14-oxPAPC complex was thencaptured using neutravidin beads. The amount CD14 retained by oxPAPC wasdetermined by Western analysis. The CD14 mutant with 26DEES29mutagenized into 26AAAA29 was designated as CD14 1R. The CD14 mutantwith 26DEES29 and 37PKPD40 mutagenized into 26AAAA29 and 37AAAA40 wasdesignated as CD14 2R. The CD14 mutant with 26DEES29, 37PKPD40, 52DVE54and 74DLGQ77 mutagenized into 26AAAA29, 37AAAA40, 52AAA54 and 74AAAA77was designated as CD14 4R. FIG. 44F presents a graph showing that the 4RCD14 mutant was not internalized in response to oxPAPC or LPStreatments. Indicated lines were treated with LPS (1 μg/ml) or oxPAPC(50 μM) for the indicated times. Surface levels of CD14 were measured byflow cytometry. Line graph represents the average and error barsrepresent the standard deviation of biological duplicates from onerepresentative experiment of three.

FIGS. 45A-45G present images showing that oxPAPC induced the activationof NLRP3 inflammasome in DCs. FIG. 45A depicts bar graphs showing IL-1βsecretion results for DCs treated with LPS alone (1 μg/ml), three dosesoxPAPC (10, 50, 120 μM) or primed with LPS for 3 hours and then treatedwith oxPAPC. For this experiment, commercially available oxPAPC and anoxPAPC enriched in PEIPC were used. 18 hours after LPS administration,secreted (left panel) and cell associated (right panel) IL-1β weremeasured by ELISA. Means and standard deviations of biologicalduplicates from one experiment representative of two are shown. FIGS.45B-45D depict bar graphs showing results for WT DC or caspase-1 KO andcaspase-1/-11 dKO DCs (FIG. 45B), ASC KO DC (FIG. 45C) and NLRP3 KO DC(FIG. 45D) treated with LPS alone (1 μg/ml), oxPAPC alone (120 μM) orprimed with LPS for 3 hours and then treated with oxPAPC. 18 hours afterLPS administration, IL-1β (left panel) and TNFα (right panel) secretionwas measured by ELISA. Means and standard deviations of two independentexperiments are shown. FIG. 45E depicts bar graphs depicting IL-1βsecretion results for MΦs treated with LPS alone (1 μg/ml), threedifferent doses oxPAPC (10, 50, 120 μM) or primed with LPS for 3 hoursand then treated with oxPAPC. For this experiment, commerciallyavailable oxPAPC and an oxPAPC enriched in PEIPC were used. 18 hoursafter LPS administration, secreted (left panel) and cell associated(right panel) IL-1β were measured by ELISA. Means and standarddeviations of biological duplicates from one experiment representativeof two are shown. FIG. 45F depicts a bar graph showing IL-1β secretionresults for MΦs treated with Pam3CSK (P3C) alone (1 μg/ml), oxPAPC alone(120 μM), ATP alone (5 mM) or primed with Pam3CSK for 3 hours and thentreated with oxPAPC, ATP, DOTAP, LPS (5 μg) or oxPAPC encapsulated inDOTAP. 18 hours after P3C administration, IL-1β was measured by ELISA.Means and standard deviations of three replicates of one experiment oftwo are shown. FIG. 45G depicts bar graphs showing that LPS-primed DCsand MΦs showed intrinsic differences in their response to NLRP3activation after ATP treatment. DCs (left panel) or MΦs (right panel)were primed with LPS (1 μg/ml) for three hours and treated with ATP (3mM). At indicated time points, IL-1β was measured by ELISA and celldeath was measured by PI permeabilization assay. Means and standarddeviations of four replicates of one representative experiment of threeare shown.

FIGS. 46A-46G show oxPAPC non-canonical inflammasome activation. FIG.46A presents a bar graph showing IL-1β secretion results for WT DC andcaspase-11 KO DC treated with LPS alone (1 μg/ml), oxPAPC alone (120 μM)or primed with LPS for 3 hours and then treated with oxPAPC. 18 hoursafter LPS administration, IL-1β secretion was measured by ELISA. Meansand standard deviations of two independent experiments are shown. FIG.46B depicts a bar graph showing TNFα secretion results for WT DC andcaspase-11 KO DC treated with LPS alone (1 μg/ml), oxPAPC alone (120 μM)or primed with LPS for 3 hours and then treated with oxPAPC. 18 hoursafter LPS administration, TNFα secretion was measured by ELISA. Meansand standard deviations of two independent experiments are shown. FIG.46C depicts images showing that LPS-primed DCs formed specks in acaspase-11-dependent manner in response to oxPAPC but not ATP. DCs wereleft untreated or primed with LPS (1 μg/ml) for three hours and thenstimulated with ATP (1 mM) or oxPAPC (120 μM). Specks containing ASC(green) and caspase-1 (Casp1, red) were analyzed 18 hours after LPSstimulation. Nuclei are shown in blue. Panels are representative of fourindependent experiments. FIG. 46D depicts blots showing that endogenouscaspase-11 associated with oxPAPC in vitro. 5100 fractions (0.5 mg) ofnontreated (nt) or P3C-primed (P3C) MΦs were incubated with biotinylatedLPS (Bio-LPS) or biotinylated-oxPAPC (Bio-oxPAPC). Endogenous proteinsassociated with biotinylated-lipids were captured by streptavidin beadsand revealed by Western analysis. Shown is a representative blot out ofthree independent experiments. FIG. 46E shows graphs of the SPR analysisof the interactions between the proteins and indicated lipids. FIG. 46Fdepicts a graph showing the gel filtration analysis of the size ofcaspase-11 complexes before and after exposure to oxPAPC. Complex sizewas monitored by A280 or Western analysis, as indicated. FIG. 46Gdepicts bar graphs showing secretion and viability results for bonemarrow cells infected with the pMSCV2.2-IRES-GFP vector (empty), thepMSCV2.2-IRES-GFP vector encoding WT caspase-11 (WT caspase-11) or thesame vector containing a catalytic mutant caspase-11 (C254A). Afterseven days of differentiation in GM-CSF containing medium, DCs wereprimed or not with LPS (1 μg/ml) for three hours and then stimulatedwith oxPAPC (120 μM), or transfected with LPS-containing FuGENE (LPS, 5μg). 18 hours after LPS priming, supernatant were collected and IL-1βand TNFα secretion was measured by ELISA. Cell viability was assessed bymeasuring LDH release.

FIGS. 47A-47E depict bar graphs showing that CD14 promoted caspase-11mediated IL-1β release from DCs. FIG. 47A depicts bar graphs showingsecretion results for WT DCs and CD14KO DCs treated with LPS alone (1μg/ml), Pam3CSK (P3C) alone (1 μg/ml), oxPAPC alone (120 μM) or primedwith LPS or with Pam3CSK for 3 hours and then treated with oxPAPC. 18hours after LPS or P3C administration, IL-1β and TNFα secretion wasmeasured by ELISA. Means and standard deviations of two independentexperiments are shown. FIG. 47B depicts bar graphs showing IL-1β andTNFα secretion results for spleen-derived WT DCs, CD14 KO DCs, andcaspase-11 KO DCs treated with LPS alone (1 μg/ml), ATP alone (1.5 mM),oxPAPC alone (120 μM) or primed with LPS for 3 hours and then treatedwith oxPAPC or ATP. 18 hours after LPS administration, IL-1β and TNFαsecretion was measured by ELISA. Means and standard deviations of tworeplicates of one representative experiment of three are shown. FIG. 47Cdepicts bar graphs showing gene expression results for WT DCs and CD14KO DCs treated with LPS or Pam3CSK. Gene expression relative to TBP wasanalyzed by qPCR at times indicated. Results are shown as geneexpression compared to untreated cells. Line graphs represent theaverage of triplicate readings from one representative experiment ofthree. FIG. 47D depicts bar graphs showing IL-1β and TNFα secretionresults for WT DC and CD14 KO DC treated with LPS alone (1 μg/ml),oxPAPC alone (120 μM) or primed with LPS for 3 hours and then treatedwith oxPAPC in the presence or the absence of rIFNβ (100 U/ml). 18 hoursafter LPS administration, IL-1β and TNFα secretion was measured byELISA. Means and standard deviations of two independent experiments areshown. FIG. 47E depicts bar graphs showing IL-1β secretion results forWT DC and CD14 KO DCs treated with LPS alone (1 μg/ml), Pam3CSK (P3C)alone (1 μg/ml), oxPAPC alone (120 μM) or primed with Pam3CSK for 3hours and then treated with oxPAPC. As indicated, LPS or oxPAPC werecomplexed with DOTAP before addition to the cell culture. Whereindicated, cells where treated with the pan-caspase inhibitor zVAD 30min before addition of the DOTAP/oxPAPC complex to the culture. 18 hoursafter stimuli administration, IL-1β secretion was measured by ELISA.Means and standard deviations of two independent experiments are shown.

FIGS. 48A-48G depict images showing that oxPPAC acted like a naturaladjuvant, preventing DC death and potentiating adaptive immuneresponses. FIG. 48A depicts a bar graph showing viability results forDCs treated with LPS alone (1 μg/ml), ATP alone (1 mM), oxPAPC alone(120 μM) or FuGENE complexed LPS (5 μg) (Fugene (LPS)), or primed forthree hours with LPS (1 μg/ml) and then treated with the indicatedstimuli. 4 and 18 hours after LPS priming, cell death was measured byLDH release. FIG. 48B depicts a bar graph showing IL-1β secretionresults for DCs treated with LPS alone (1 μg/ml), ATP alone (1 mM),oxPAPC alone (120 μM) or FuGENE complexed LPS (5 μg) (Fugene (LPS)), orprimed for three hours with LPS (1 μg/ml) and then treated with theindicated stimuli. 4 and 18 hours after LPS priming, IL-1β secretion wasmeasured by ELISA. FIGS. 48C-48D depict images showing staining resultsfor DCs pretreated with LPS for 3 hours (1 μg/ml) and then activatedwith ATP (1 mM) or oxPAPC (120 μM). 18 hours later, cells were stainedfor ASC (green), nuclei (blue) Zombie Dye (red) (FIG. 48C) or activemitochondria (red) (FIG. 48D). Panels are representative of threeindependent experiments. FIGS. 48E-48F depict bar graphs showingviability results for DCs treated with LPS alone (1 μg/ml), oxPAPC alone(120 μM), ATP alone (1 mM) or primed with LPS for 3 hours and thentreated with oxPAPC or ATP. At the indicated time points, cell viabilitywas measured by 7-AAD staining. Means and standard deviations of twoindependent experiments are shown. FIG. 48G depicts bar graphs showingthat oxPAPC potentiated memory T cell responses in vivo. CD4+ T-cellswere isolated from the draining lymph nodes 40 days after immunizationwith OVA+LPS in IFA (LPS), OVA+LPS+oxPAPC in IFA (LPS+oxPAPC) orOVA+oxPAPC in IFA (oxPAPC) of WT, caspase-1/-11 dKO or caspase-11 KOmice. CD4+ T-cells were restimulated or not with OVA in the presence ofDCs. IFNγ (left panel) and IL-17 (right panel) secretion was measured 5days later by ELISA. Bar graphs represent means and standard errors oftwo experiments with five animals per group.

FIGS. 49A-49B depict blots showing that oxPAPC did not induce myddosomeformation or type I IFN signaling (related to FIGS. 43A-43D). FIG. 49Apresents a blot showing myddosome formation in assessed at the indicatedtime points after treatment with LPS (1 μg/ml) or different doses ofoxPAPC (10, 50, 120 μM) by co-immunoprecipitation of IRAK4 with MyD88followed by Western analysis for the proteins indicated. FIG. 49Bdepicts a blot showing whole cell lysates (WCL) collected from DCs andmonitored by Western analysis for the proteins indicated after treatmentwith LPS (1 μg/ml) or different doses of oxPAPC (10, 50, 120 μM).

FIGS. 50A-50D depict graphs showing that oxPAPC was an agonist for CD14but not for TLR4 (see also FIGS. 44A-44F). FIG. 50A depicts a bar graphshowing surface CD14 results for DCs treated with oxPAPC at theindicated concentrations. Surface levels of CD14 were measured by flowcytometry. Line graphs represent means and standard deviations of twoindependent experiments. FIGS. 50B-50D depict bar graphs showing surfaceCD14, TLR4 and TLR4 dimerization results for DCs treated with LPS (1μg/ml) or oxPAPC (120 μM) for the indicated times in the presence orabsence of cycloheximide (100 μg/ml). Surface levels of CD14 (FIG. 50B),TLR4 (FIG. 50C) and TLR4 dimerization (FIG. 50D) were measured by flowcytometry. Line graphs represent means and standard deviations of twoindependent experiments.

FIGS. 51A-51I depict images showing that oxPAPC induced IL-1β release ina cell-type specific manner (see also FIGS. 45A-G). FIG. 51A depicts abar graph showing that DCs and released IL-1β in response to ATP.Freshly derived DCs and MΦs were treated with LPS alone (1 μg/ml), ATPalone (ATPlow: 0.5 mM; ATPhi: 5 mM) or were primed with LPS for 3 hoursand then treated with ATP. Supernatants were collected 18 hours afterLPS administration and levels of secreted IL-1β were measured by ELISA.Means and standard deviations of two independent experiments are shown.FIGS. 51B-51C depict bar graphs showing secreted and cell-associatedIL-1β levels for DCs primed or not primed with different doses of LPS(100 and 1000 ng/ml) and 3 hours later cells were activated with oxPAPC(120 μM) or ATP (0.5 mM). Supernatants were collected 18 hours after LPSadministration and levels of secreted IL-1β (FIG. 51B) and cellassociated IL-1β (FIG. 51C) were measured by ELISA. Means and standarddeviations of biological duplicates from one experiment representativeof three are shown. FIG. 51D depicts a bar graph showing TNFα resultsfor DCs or MΦs treated with LPS alone (1 μg/ml), ATP alone (0.5 mM),oxPAPC alone (120 μM) or primed with LPS for 3 hours and then treatedwith ATP or oxPAPC. Supernatants were collected 18 hours after LPSadministration and levels of TNFα were measured by ELISA. Means andstandard deviations of two independent experiments are shown. FIG. 51Edepicts a bar graph showing secreted and cell-associated IL-1β resultsfor DCs treated with LPS alone (1 μg/ml), ATP alone (0.5 mM), oxPAPCalone (120 μM) or co-administered with LPS and ATP or LPS and oxPAPC.Secreted and cell-associated IL-1β were measured by ELISA 18 hourslater. Means and standard deviations of two independent experiments areshown. FIG. 51F depicts a bar graph showing IL-1β and TNFα secretionresults for DCs treated with LPS alone (1 μg/ml), ATP alone (5 mM), theindicated phospholipids alone (120 μM) or primed with LPS for 3 hoursand then treated with ATP or the indicated phospholipids. 18 hours afterLPS administration, IL-1β and TNFα secretion was measured by ELISA.Means and standard deviations of two independent experiments are shown.FIG. 51G depicts bar graphs showing IL-1β and TNFα secretion results forDCs and MΦs (pre-treated or not with IFNγ) treated with LPS alone (1μg/ml), ATP alone (1 mM for DCs and 5 mM for MΦs), oxPAPC alone (120 μM)or pretreated with LPS for three hours and then activated with ATP oroxPAPC. Secreted IL-1β and TNFα were measured by ELISA 18 hours later.Means and standard deviations of two replicates of one representativeexperiment of two are shown. FIG. 51H depicts a blot showing that DCsand MΦs expressed different levels of ASC protein. DCs and MΦs weretreated or not with LPS for 4 hours. Total lysates were used to assessthe protein abundance of ASC by Western analysis. FIG. 51I depicts bargraphs showing gene expression results for DCs and MΦs treated or nottreated with LPS for 4 hours. ASC, Nlrp3, caspase-1 (Casp-1) andcaspase-11 (Casp-11) expression was measured by qPCR. Gene expressionlevels relative to TBP are shown. Line graphs represent the average oftriplicate readings from one representative experiment of three.

FIGS. 52A-52I depict images showing that oxPAPC bound to caspase-11 andregulated inflammasome activation in DCs (see also FIGS. 46A-46G). FIG.52A depicts a line graph showing ASC and caspase-1 containing speckformation for DCs primed with LPS (1 μg/ml) and then activated with ATP(1 mM) or oxPAPC (120 μM). At the indicated times, ASC and caspase-1containing speck formation was assessed. Data represent means andstandard deviations of three fields containing around 50 cells obtainedin three independent experiments. FIG. 52B depicts a bar graph showingASC and caspase-1 containing speck formation for DCs primed with LPS (1μg/ml) and then activated with ATP (1 mM) or oxPAPC (120 μM). 18 hourslater ASC and caspase-1 containing speck formation was assessed. Datarepresent means and standard deviations of three independentexperiments. FIG. 52C depicts bar graphs showing IL-1β and TNFαsecretion for DCs of the indicated genotype treated with Pam3CSK (P3C)alone (1 μg/ml), ATP alone (0.5 mM), oxPAPC alone (120 μM) or primedwith Pam3CSK for 3 hours and then treated with ATP or oxPAPC. 18 hoursafter Pam3CSK administration, IL-1β and TNFα secretion was measured byELISA. Means and standard deviations of two independent experiments areshown. FIG. 52D depicts a bar graph showing IL-1β and TNFα secretion forDCs treated with CpG alone (1 μM), oxPAPC alone (120 μM) or primed withCpG for 3 hours and then treated with oxPAPC. 18 hours after CpGadministration, IL-1β and TNFα secretion was measured by ELISA. Meansand standard deviations of two independent experiments are shown. FIG.52E depicts bar graphs showing secreted and cell-associated IL-1β levelsfor DCs derived from C3H/HeSNJ (WT C3H) and C3H/HeJ (TLR4 mutant C3H)mice treated with Pam3CSK (P3C) alone (1 μg/ml), oxPAPC alone (120 μM)or primed with Pam3CSK for 3 hours and then treated with oxPAPC. 18hours after LPS administration, secreted (left panel) and cellassociated (right panel) IL-1β were measured by ELISA. Means andstandard deviations of biological duplicates from one experimentrepresentative of two are shown. FIG. 52F depicts a line graph showingamounts of infectious virus in the eye for WT C57BL/6 (WT) or caspase-11KO mice infected with HSV-1 in the eye. At the indicated times, theamount of infectious virus in the eye was measured by plaque assay. FIG.52G depicts blots showing Caspase-11 levels in lysates from 293T cellsexpressing indicated caspase-11 alleles incubated with biotinylatedligands and streptavidin beads. Caspase-11 retained by the biotinylatedligands and inputs were detected by western analysis. Shown is arepresentative blot out of three independent experiments. Alleles ofcaspase-11 used were as follows: caspase-11 (WT), the CARD domain (1-92a.a.) and the delta CARD (ACARD) domain (93-373 a.a.). FIGS. 52H-52Idepict graphs showing enzymatic activity for recombinant caspase-11monomers or oligomers mixed with the lipids indicated. Enzymaticactivity was monitored over time by spectrofluorimetry.

FIGS. 53A-53D depict images showing that the inflammasome componentexpression was similar in WT DC and CD14KO DCs (Related to FIGS.47A-47E). FIG. 53A depicts a bar graph showing IL-18 levels in WT DCs,CD14KO DCs and caspase-11 KO DCs treated with LPS alone (1 μg/ml),oxPAPC alone (120 μM), ATP alone (1 mM) or primed with LPS for 3 hoursand then treated with oxPAPC or ATP. IL-18 was measured by ELISA in thesupernatant 18 hours later. FIG. 53B depicts flow cytometry data showingMHC class II and CD40 levels for splenic DCs treated with LPS alone (1μg/ml), oxPAPC alone (120 μM), or primed with LPS for 3 hours and thentreated with oxPAPC. 18 hours later, MHC class II and CD40 levels weremeasured by flow cytometry. FIG. 53C depicts bar graphs showing geneexpression results for WT DC and CD14 KO DC treated with LPS or Pam3CSK(1 μg/ml). Gene expression relative to TBP was analyzed by qPCR at timesindicated. Untreated cells were used as a negative control in allexperiments. FIG. 53D depicts bar graphs showing Secreted andcell-associated IL-1β for DCs pre-treated or not for 30 min withchloroquine (10 μM) and then stimulated with LPS alone (1 μg/ml), oxPAPCalone (120 μM) or primed with LPS and then treated with oxPAPC. Secretedand cell-associated IL-1β were measured by ELISA 18 hours later.

FIGS. 54A-54C depict images showing that oxPAPC did not inducepyroptosis in DCs and promoted enhanced T-cell activation (see alsoFIGS. 48A-48G). FIG. 54A depicts a line graph showing pyroptotic celllevels for WT DCs treated with LPS alone (1 μg/ml), or primed with LPSfor 3 hours and then treated with oxPAPC (120 μM) or ATP (5 mM).Pyroptosis induction was assessed up to 18 hours after stimuli addition.Pyroptotic cells were identified as Annexin V and 7-AAD double positivecells by flow cytometry. Data are representative of two independentexperiments. FIG. 54B depicts a bar graph showing IL-2 secretion forCD4+ T-cells isolated from the draining lymph nodes 40 days afterimmunization with OVA+LPS in IFA (LPS), OVA+LPS+oxPAPC in IFA(LPS+oxPAPC) or OVA+oxPAPC in IFA (oxPAPC) of WT, caspase-1/-11 dKO orcaspase-11 KO mice. CD4+ T-cells were restimulated or not with OVA inthe presence of DC as antigen presenting cells. IL-2 secretion wasmeasured 5 days later by ELISA. Bar graphs represent means and standarderrors of two experiments with five animals per group. FIG. 54C depictsbar graphs showing that oxPAPC potentiated effector T cell responses invivo. CD4+ T-cells were isolated from the draining lymph nodes 7 daysafter immunization with OVA+LPS in IFA (LPS), OVA+LPS+oxPAPC in IFA(LPS+oxPAPC) or OVA+oxPAPC in IFA (oxPAPC) of WT, caspase-1/-11 dKO orcaspase-11 KO mice. CD4+ T-cells were restimulated or not with OVA inthe presence of DC as antigen presenting cells. IFNγ (upper panel) andIL-17 (lower panel) secretion was measured 5 days later by ELISA. Bargraphs represent means and standard errors of four experiments withthree mice per group.

DETAILED DESCRIPTION

The present invention relates, at least in part, to the unexpectedobservation that a PAPC lipid, particularly oxidized PAPC lipids(oxPAPCs), functions as a specific activator of inflammatory responsesin dendritic cells (DCs). Specifically,1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and itsoxidized variant (oxPAPC) were identified as the first specificactivators of inflammatory responses in dendritic cells (DCs). In thepresence of TLR ligands, oxPAPC promoted DC survival and triggered therelease of the T-cell activating cytokine, interleukin 1 beta (IL-1β).

Without wishing to be bound by theory, mechanistically, oxPAPC isbelieved to have engaged the LPS receptor CD14 on the surface of DCs,which promoted its delivery into endosomes and subsequent access to thecytosolic protein caspase-11. Caspase-11 engagement by oxPAPC triggeredthe inflammasome-mediated release of IL-1β. Remarkably, these oxPAPCtriggered responses did not occur in macrophages, indicating that theactions of this lipid were uniquely designed to promoteimmuno-regulatory activities of DCs, as opposed to general(macrophage-mediated) inflammatory responses. Consequently, oxPAPCsynergized with microbial products to induce a more robust activation ofantigen specific T-cells than could be elicited by PAMPs alone. oxPAPCwas therefore identified as a member of a new class of immuno-modulatoryfactors (termed “vita-DAMPs”), which function together with PAMPs topromote DC survival and elicit maximal adaptive immune responses.

DCs are the most potent activators of protective (adaptive) immunity,and much current work is focused on designing vaccine adjuvants thatselectively promote DC-mediated immunity. All currently FDA-approvedvaccine adjuvants are unable to specifically activate DCs. They allpromote general inflammatory responses in a variety of immune cells,including macrophages, DCs and others.

The current discovery that PAPCs can specifically activate functions inDCs has identified this molecule as a lead candidate for anext-generation vaccine adjuvant. Of note, PAPCs have been studied inthe past by other research groups, but most research in this area hasfocused on their ability to act as anti-inflammatory molecules. Thecurrent discovery that PAPCs act to promote immunity, rather thaninhibit immunity, distinguishes the currently described and exemplifieduses of PAPCs from previous suggestions regarding the therapeutic valueof these molecules.

A key finding in identifying the current invention was the discoverythat PAPCs were only capable of promoting DC-mediated immune responsesif co-administered with microbial products. This co-administrationcreated a state of the DCs that had not been observed before, and it isforeseen that this novel cellular behavior is of prime therapeuticpotential.

Definitions

The term “oxPAPC” or “oxidized PAPC”, as used herein, refers to lipidsgenerated by the oxidation of1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), whichresults in a mixture of oxidized phospholipids containing eitherfragmented or full length oxygenated sn-2 residues. Well-characterizedoxidatively fragmented species contain a five-carbon sn-2 residuebearing omega-aldehyde or omega-carboxyl groups. Oxidation ofarachidonic acid residue also produces phospholipids containingesterified isoprostanes. oxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC andKOOA-PC species, among other oxidized products present in oxPAPC.

The term “non-canonical inflammasome-activating lipid”, as used herein,refers to a lipid capable of eliciting an inflammatory response in acaspase 11-dependent inflammasome of a cell. Exemplary “non-canonicalinflammasome-activating lipids” include PAPC, oxPAPC and species ofoxPAPC (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC), as well as RhodoLPS (LPS-RS or LPS from Rhodobacter sphaeroides).

“Immunogen” and “antigen” are used interchangeably and mean any compoundto which a cellular or humoral immune response is to be directedagainst. Non-living immunogens include, e.g., killed immunogens, subunitvaccines, recombinant proteins or peptides or the like. The adjuvants ofthe invention can be used with any suitable immunogen. Exemplaryimmunogens of interest include those constituting or derived from avirus, a mycoplasma, a parasite, a protozoan, a prion or the like.Accordingly, an immunogen of interest can be from, without limitation, ahuman papilloma virus, a herpes virus such as herpes simplex or herpeszoster, a retrovirus such as human immunodeficiency virus 1 or 2, ahepatitis virus, an influenza virus, a rhinovirus, respiratory syncytialvirus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, a bacteriumof the genus Salmonella, Staphylococcus, Streptococcus, Enterococcus,Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, amalarial parasite, and/or Trypanosoma cruzi. It is further contemplatedthat adjuvant lipids of the invention can be co-administered with tumoror other cancer antigens, thereby providing an immunostimulatory cancertherapy/cancer vaccine.

“Concurrently administered” as used herein means that two compounds areadministered sufficiently close in time to achieve a combinedimmunological effect. Concurrent administration may thus be carried outby sequential administration or simultaneous administration (e.g.,simultaneous administration in a common, or the same, carrier).

The “modulation” of, e.g., a symptom, level or biological activity of amolecule, or the like, refers, for example, to the symptom or activity,or the like that is detectably increased or decreased. Such increase ordecrease may be observed in treated subjects as compared to subjects nottreated with an adjuvant lipid of the invention (a non-canonicalinflammasome-activating lipid), where the untreated subjects (e.g.,subjects administered immunogen in the absence of adjuvant lipid) have,or are subject to developing, the same or similar disease or infectionas treated subjects. Such increases or decreases may be at least about2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more orwithin any range between any two of these values. Modulation may bedetermined subjectively or objectively, e.g., by the subject'sself-assessment, by a clinician's assessment or by conducting anappropriate assay or measurement, including, e.g., assessment of theextent and/or quality of immunostimulation in a subject achieved by anadministered immunogen in the presence of an adjuvant lipid of theinvention (a non-canonical inflammasome-activating lipid). Modulationmay be transient, prolonged or permanent or it may be variable atrelevant times during or after an adjuvant lipid of the invention isadministered to a subject or is used in an assay or other methoddescribed herein or a cited reference, e.g., within times describedinfra, or about 12 hours to 24 or 48 hours after the administration oruse of an adjuvant lipid of the invention to about 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more after asubject(s) has received such an immunostimulatory composition/treatment.

As used herein, “subject” includes animals that possess an adaptiveimmune system, as described herein, such as human (e.g., human subjects)and non-human animals. The term “non-human animals” includes allvertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals,such as non-human primates, e.g., sheep, dog, cow, chickens, amphibians,reptiles, etc.

A “suitable dosage level” refers to a dosage level that provides atherapeutically reasonable balance between pharmacological effectivenessand deleterious effects (e.g., sufficiently immunostimulatory activityimparted by an administered immunogen in the presence of an adjuvantlipid of the invention, with sufficiently low macrophage stimulationlevels). For example, this dosage level can be related to the peak oraverage serum levels in a subject of, e.g., an anti-immunogen antibodyproduced following administration of an immunogenic composition(comprising an adjuvant lipid of the invention) at the particular dosagelevel.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Regulation of Dendritic Cells (DCs) and Pattern Recognition Receptors(PRRs)

The innate immune system has classically been viewed to operate in anall-or-none fashion, with DCs operating either to mount inflammatoryresponses that promote adaptive immunity, or not. TLRs expressed by DCsare therefore believed to be of central importance in determiningimmunogenic potential of these cells. The mammalian immune system isresponsible for detecting microorganisms and activating protectiveresponses that restrict infection. Central to this task are thedendritic cells, which sense microbes and subsequently promote T-cellactivation. It has been suggested that dendritic cells can gauge thethreat of any infection and instruct a proportional response (Blander,J. M. (2014). Nat Rev Immunol 14, 601-618; Vance, R. E. et al., (2009)Cell host & microbe 6, 10-21), but the mechanisms by which theseimmuno-regulatory activities could occur are unclear.

PRRs act to either directly or indirectly detect molecules that arecommon to broad classes of microbes. These molecules are classicallyreferred to as pathogen associated molecular patterns (PAMPs), andinclude factors such as bacterial lipopolysaccharides (LPS), bacterialflagellin or viral double stranded RNA, among others.

An important attribute of PRRs as regulators of immunity is theirability to recognize specific microbial products. As such, PRR-mediatedsignaling events should provide a definitive indication of infection. Itwas postulated that a “GO” signal is activated by PRRs expressed on DCsthat promote inflammation and T-cell mediated immunity. Interestingly,several groups have recently proposed that DCs may not simply operate inthis all-or-none fashion (Blander, J. M., and Sander, L. E. (2012). NatRev Immunol 12, 215-225; Vance, R. E. et al., (2009) Cell host & microbe6, 10-21). Rather, DCs may have the ability to gauge the threat (orvirulence) that any possible infection poses and mount a proportionalresponse. The most commonly discussed means by which virulence can begauged is based on the ability of virulent pathogens to activate agreater diversity of PRRs than non-pathogens. However, not all microbeshave a common set of PRR activators, and not all PRR activators are ofcomparable potency. The number of PRRs activated during an infection maytherefore not be an ideal gauge of virulence. Moreover, increasing thenumber of PRRs activated during an infection will lead to a greaterinflammatory response in general, which may indirectly promote greaterT-cell responses. Conditions previously suggested to heighten the stateof DC activation (e.g. through the use of virulent pathogens as stimuli)are also expected to heighten the state of MΦ activation (Vance, R. E.et al., (2009) Cell host & microbe 6, 10-21). Thus, it remains unclearif mechanisms are truly in place for the immune system (i.e. DCs) togauge the threat of an infection specifically.

One possible means by which the threat of infection could be assessedwould be through the well-recognized process of coincidence detection,where independent inputs result in a response that differs from the oneelicited by any single input. In the context of PRRs, one such inputmust be a microbial product as an indicator of infection, regardless ofthe threat of virulence. In order to gauge the virulence threat, asecond input must exist. Without wishing to be bound by theory, it isnow thought that this putative second input is a molecule produced atthe site of tissue injury, as cellular damage is often a featureassociated with highly pathogenic microbes. Candidate molecules that mayprovide a second stimulus to DCs are the diverse family of moleculescalled danger associated molecules patterns (DAMPs), which are alsoknown as alarmins (Kono, H., and Rock, K. L. (2008) Nat Rev Immunol 8,279-289; Pradeu, T., and Cooper, E. L. (2012) Front Immunol 3, 287).DAMPs have been found at sites of infectious and non-infectious tissueinjury, and have been proposed to modulate inflammatory responses,although their mechanisms of action remain unclear. One such class ofDAMPs is represented by oxidized phospholipids derived from1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), whichare collectively known as oxPAPC. These lipids are produced at the sitesof both infectious and non-infectious tissue injury (Berliner, J. A.,and Watson, A. D. (2005). N Engl J Med 353, 9-11; Imai, Y. et al. (2008)Cell/33, 235-249; Shirey, K. A. et al. (2013) Nature 497, 498-502) andare found at very high levels in the membranes of dying cells (Chang, M.K. et al., (2004) J Exp Med 200, 1359-1370). oxPAPC is also an activecomponent of oxidized low density lipoprotein (oxLDL) aggregates thatpromote inflammation in atherosclerotic tissues (Leitinger, N. (2003)Curr Opin Lipidol 14, 421-430), where local concentrations can be ashigh as 10-100 μM (Oskolkova, O. V. et al. (2010) J Immunol 185,7706-7712). The association between oxPAPC and dying cells raised thepossibility that these lipids could serve as a generic indicator oftissue health. In the presence of microbial product(s), oxPAPC maytherefore indicate an increased infectious threat.

Without wishing to be bound by theory, mechanistically, the receptorCD14 appears to capture lipids such as oxPAPC and PAPC and deliver themto an intracellular location where they activate non-canonicalinflammasomes (Caspase 11-dependent inflammasomes).Inflammasome-mediated activities then synergize with independentlyoccurring TLR signaling events to promote more robust T-cell responsesthan those induced by TLR ligands alone. Thus, oxidized lipids appear toalert dendritic cells to a highly infectious microbial encounter,allowing these cells to promote an adaptive response that iscommensurate with the infectious threat.

Toll-Like Receptors

Toll-like receptors (TLRs) are type I transmembrane receptors,evolutionarily conserved between insects and humans. Ten TLRs have sofar been established (TLRs 1-10) (Sabroe, I. et al., (2003) Journal ofImmunology 171(4): 1630-5). Members of the TLR family have similarextracellular and intracellular domains; their extracellular domainshave been shown to have leucine-rich repeating sequences, and theirintracellular domains are similar to the intracellular region of theinterleukin-1 receptor (IL-1 R). TLR cells are expressed differentiallyamong immune cells and other cells (including vascular epithelial cells,adipocytes, cardiac myocytes and intestinal epithelial cells). Theintracellular domain of the TLRs can interact with the adaptor proteinMyd88, which also posses the IL-1 R domain in its cytoplasmic region,leading to NF-KB activation of cytokines; this Myd88 pathway is one wayby which cytokine release is effected by TLR activation. The mainexpression of TLRs is in cell types such as antigen presenting cells(e.g. dendritic cells, macrophages etc.). One such TLR is TLR4, which isresponsible for activating the innate immune system and recognizeslipopolysaccharide (LPS), a component of gram-negative bacteria. TLR4has been shown to interact with lymphocyte antigen 96, Myd88 (myeloiddifferentiation primary response gene 88), and TOLLIP (toll interactingprotein).

Activation of dendritic cells by stimulation through the TLRs leads tomaturation of dendritic cells, and production of inflammatory cytokinessuch as IL-12. Research carried out so far has found that TLRs recognizedifferent types of agonists, although some agonists are common toseveral TLRs. TLR agonists are predominantly derived from bacteria orviruses, and include molecules such as flagellin or bacteriallipopolysaccharide (LPS).

Two States of DC Activation

Two states of DC activation were identified herein. The first activationstate was mediated by encounters with microbial products, such as TLRligands. These ligands activated TLRs to release cytokines, upregulateco-stimulatory molecules and promote MHC-mediated antigen presentation,all of which were important for T-cell activation. However, these TLRligands were common to pathogens and non-pathogens and cannot be used togauge threat to the host. The second state of DCs was considered“hyperactive”, and was mediated by coincident encounters with microbialproducts and oxidized phospholipids that were abundant at sites oftissue damage. The coincident detection of TLR ligands and oxidizedlipids (e.g. oxPAPC) promoted all the activities elicited by theclassical activation state, and induced the inflammasome-mediatedrelease of IL-1β, a potent activator of T-cells. Neither TLR ligandsalone nor oxPAPC alone possessed the ability to induce IL-1β release, anobservation that provided formal experimental evidence that the innateimmune system employed the principle of coincidence detection to inducea hyperactive state in DCs.

An intriguing aspect of the hyperactive DC state was the mechanism bywhich it was elicited. Whereas the classical activation was elicited bymicrobial products, the hyperactive state was elicited by microbialproducts and self-derived products. This self-referential aspect ofimmune activation was not without precedent, as T-cell maturation andmaintenance had been previously shown to rely upon interactions with MHCmolecules bearing microbial and self-peptides (Janeway, C. A., Jr.(2002) Annu Rev Immunol 20, 1-28). Mechanistically, the analysis ofoxPAPC revealed this molecule to be a selective endogenous mimic of LPS,in that it bound and activated the LPS receptors CD14 and caspase-11.Interestingly, oxPAPC did not induce TLR4 dimerization, endocytosis,myddosome formation or gene expression. In fact, when administered priorto microbial encounters, oxPAPC acted as a TLR4 antagonist (Bochkov, V.N. et al., (2002) Nature 419, 77-81; Erridge, C. et al., (2008) TheJournal of biological chemistry 283, 24748-24759; Oskolkova, O. V. etal. (2010) J Immunol 185, 7706-7712). The collective data thereforerevealed an intriguing cellular process by which CD14 functions tocoordinate the activities of TLR4 and caspase-11, by delivering eitherPAMPs (LPS) or DAMPs (oxPAPC) to their respective receptors.

Several observations supported the central aspect of this proposedCD14-caspase-11 pathway to DC hyperactivation. First, oxPAPC formed acomplex with CD14 and caspase-11 in vitro. Second, genetic deficienciesof CD14 and caspase-11 phenocopied one another, in that the loss ofeither protein resulted in an inability of DCs to release IL-1β inresponse to oxPAPC treatment. In contrast, neither CD14 nor caspase-11was required for ATP-mediated IL-1β release. Third, neither of theseproteins was required during the priming phase of inflammasomeactivation, as assessed by the normal level of expression of variousTLR-dependent cytokines. Fourth, binding of oxPAPC to CD14 promotedendocytosis and the delivery of this lipid to intracellular caspase-11.Support for this statement derived from the ability of transfection ofoxPAPC into the cytosol to rescue defects in IL-1β release in CD14 KOs.This observation provided definitive evidence that the transportfunction of CD14 was critical for caspase-11 activation.

Caspase-11 has attracted much attention in recent years, based on itsability to promote IL-1β release and pyroptosis in response togram-negative cytosolic bacteria (Hagar, J. A. et al., (2013) Science341, 12501253; Kayagaki, N. et al. (2013) Science 341, 1246-1249). Thisselectivity of caspase-11 in promoting immune responses to gram-negativebacteria was explained by its newly recognized ability to operate as abona fide LPS receptor (Shi, J. et al., (2014a) Nature 514, 187-192).oxPAPC bound to caspase-11 and extended the role of caspase-11 beyondits operation as an LPS receptor. Indeed, caspase-11 was required foroxPAPC-mediated IL-1β release when TLR4 ligands were not present, suchas when cells were primed with ligands often associated withgram-positive bacteria (i.e. Pam3CSK) or viruses (CpG DNA).

Based on these data, caspase-11 had a general function as a gauge ofvirulence threat in DCs. The threat was assessed in two ways. First, byvirtue of its ability to bind oxPAPC, a self-derived indicator ofdamage, caspase-11 may become activated during encounters with anypathogen that causes tissue damage and cell death. This activity wouldtherefore provide DCs with a general mechanism to become hyperactivatedduring infections with virulent microorganisms. Secondly, in the case ofbacteria that encode Type III and Type IV secretion systems, whichdeliver LPS to the cytosol directly (Hagar, J. A., and Miao, E. A.(2014) Curr Opin Microbiol 17, 61-66), caspase-11 likely hyperactivatedDCs even before tissue damage occurred. Under these latter conditions,the delivery of LPS to the cytosol by virulence-associated secretionsystems should not have involved the transport functions of CD14.Indeed, it was identified herein that the genetic requirement of CD14for inflammasome activation could be bypassed by transfection of LPS oroxPAPC directly into the cytosol. Natural delivery of oxPAPC from theextracellular media to caspase-11, in contrast, was dependent upon CD14.This critical role of CD14 in mediating caspase-11 activation suggestedthat this protein possessed a broader function in inducing adaptiveimmunity than would be predicted by its assignment as an LPS receptor.Rather, CD14 and caspase-11 are general regulators of immunity against awide range of pathogens. In vivo support for this model came from thefinding that HSV-1 replication in the eye was restricted by the actionsof caspase-11.

The mechanistic studies also revealed that oxPAPC differed from LPS inits actions towards caspase-11 in several fundamental ways. First,whereas both lipids bound to caspase-11 and induced its oligomerization,LPS bound the CARD, whereas oxPAPC bound the catalytic domain. Thesedifferential mechanisms of engagement had functional consequences, asbinding to the CARD promoted the caspase-11 enzymatic activity, whereasbinding the catalytic domain prevented enzymatic activity. Since theenzymatic activity of caspase-11 was necessary for pyroptosis, it stoodto reason that oxPAPC should not kill cells. Indeed, throughpopulation-based and single cell analyses, oxPAPC was identified as notkilling cells, and it was identified that inflammasomes were presentwithin living DCs that have been exposed to oxPAPC. In contrast, in DCsthat were exposed to ATP, inflammasomes were present only within deadcells. In fact, oxPAPC promoted the viability of DCs that were alsoexposed to LPS. Whereas there were several examples of endogenousmolecules that bind to PRRs, most current information suggested themodes of interactions were similar to those that mediate microbialinteractions (or are unknown). Caspase-11 therefore represented anunusual PRR in that it contained distinct domains that interact withPAMPs (LPS) and endogenous molecules (oxPAPC). These distinct modes ofinteraction resulted in different cellular responses, which indicatedthat like DCs, PRRs also possessed different states of activation.

The dual activities of oxPAPC to promote inflammasome activation and DCsurvival indicated a role for these activities in the potentiation ofadaptive immune responses. Indeed, LPS/oxPAPC was identified herein as asuperior adjuvant than LPS alone, in terms of eliciting antigen-specificeffector and memory T-cells in vivo. Other instances of inflammasomeactivation occurring independent of cell death have also been observed(Broz, P. et al., (2010) Cell Host Microbe 8, 471-483; Ceballos-Olvera,I. et al., (2011) PLoS Pathog 7, e1002452; Schmidt, R. L., and Lenz, L.L. (2012) PLoS One 7, e45186). Ongoing studies are exploring themechanisms by which death and IL-1β release are coordinated. Based onits unusual ability to be both a pro-inflammasome and pro-survivalstimulus, it is herein concluded that oxPAPC can be considered avita-DAMP, that functions to promote DC survival and the initiation ofadaptive immunity. vita-DAMPs can be operationally distinguished fromtraditionally-defined DAMPs, such as ATP, because they promote cellviability, as opposed to promoting pyroptotic cell death. It is alsocontemplated herein that other known TLR4 antagonists can be selectiveLPS mimics that possess similar activities as oxPAPC. It was also notedthat oxPAPC was released under non-infectious circumstances. Under theseconditions, the ability of oxPAPC to promote CD14 endocytosis likelyhelped limit TLR4-dependent inflammatory responses that could mistakenlyhave been activated by other DAMPs present at the sites of injury(Mancek-Keber, M., et al. (2015) Science signaling 8, ra60). Thiscontext-dependent activity of oxPAPC as either inhibiting or promotinginflammation is identified as critical in helping DCs gauge the sourceof damage in a given tissue.

In summary, a means by which an endogenous self-molecule can create ahyperactive state of DCs through the ability to engage caspase-11 in anatypical manner was identified herein. The existence of this hyperactivestate revealed that the innate immune system operated through amechanism by which infectious threat was assessed by the coincidentdetection of PAMPs and vita-DAMPS.

Adjuvants and Vaccines

Immunogenic compositions comprising adjuvants of the invention may beadministered to a subject using any known form of vaccine, e.g.,attenuated virus, protein, nucleic acid, etc. vaccine, so as to producein the subject, an amount of the selected immunogen which is effectivein inducing a therapeutic or prophylactic immune response against thetarget antigen in the subject. The subject may be a human or nonhumansubject. Animal subjects include, without limitation, non-humanprimates, dogs, cats, equines (horses), ruminants (e.g., sheep, goats,cattle, camels, alpacas, llamas, deer), pigs, birds (e.g., chicken,turkey quail), rodents, and chirodoptera. Subjects can be treated forany purpose, including without limitation, eliciting a protective immuneresponse, or producing antibodies (or B cells) for collection and usefor other purposes.

In certain embodiments, the invention features an adjuvant-containingmicrobial vaccine. Microbial vaccines are often comprised of the cellwall components that allow the immune system to recognize the wholeorganism, or in bacteria that cause disease through toxicity—such asDiphtheria, the toxin or derived toxoid may be used. Antitoxins arebeing developed for some disease organisms, predominantly fortherapeutic use. Bacteria may be cultured in liquid media, or as solidsubstrate cultures, harvested, purified and used directly as killed orattenuated vaccines.

Optionally, an immunogen of interest is expressed by diseased targetcells (e.g., neoplastic cell, infected cells), and expressed in loweramounts or not at all in other tissue. Examples of target cells includecells from a neoplastic disease, including but not limited to sarcoma,lymphoma, leukemia, a carcinoma, melanoma, carcinoma of the breast,carcinoma of the prostate, ovarian carcinoma, carcinoma of the cervix,colon carcinoma, carcinoma of the lung, glioblastoma, and astrocytoma.Alternatively, the target cell can be infected by, for example, a virus,a mycoplasma, a parasite, a protozoan, a prion and the like.Accordingly, an immunogen of interest can be from, without limitation, ahuman papilloma virus (see below), a herpes virus such as herpes simplexor herpes zoster, a retrovirus such as human immunodeficiency virus 1 or2, a hepatitis virus, an influenza virus, a rhinovirus, respiratorysyncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, abacterium of the genus Salmonella, Staphylococcus, Streptococcus,Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio,Mycobacterium, amoeba, a malarial parasite, and Trypanosoma cruzi.

In addition to tumor antigens and antigens of infectious agents, mutantsof tumor suppressor gene products including, but not limited to, p53,BRCA1, BRCA2, retinoblastoma, and TSG101, or oncogene products such as,without limitation, RAS, W T, MYC, ERK, and TRK, may also provide targetantigens to be used according to the invention. The target antigen canbe a self-antigen, for example one associated with a cancer orneoplastic disease. In an embodiment of the invention, the immunogen isa peptide from a heat shock protein (hsp)-peptide complex of a diseasedcell, or the hsp-peptide complex itself.

In certain embodiments, the immunogen may be purified from a naturalsource, obtained by means of recombinant expression, or synthesizeddirectly. In certain embodiments, the immunogen can be provided by wholecells, micro-organisms, or viral particles, which may be live,attenuated, or killed. In other embodiments, the immunogen may comprisea protein fragment comprising one or more immunogenic regions of themolecule.

Immunogens include those that are modified or derivatized, such as byconjugation or coupling to one or more groups to enhance an immuneresponse of the subject. Examples of immunogenic carrier proteins areKLH and BSA. Immunogenic carriers also include polypeptides that arepromiscuous Class II activators (see, e.g., Panina-Bordignon et al, ColdSpring Harb Symp Quant Biol 1989). Conjugate linkages are made bymethods well known to those of skill in the art.

Immunogenic compositions of the invention comprise an immunogen and anadjuvant lipid, and can be administered for therapeutic and/orprophylactic purposes. In therapeutic applications, an immunogeniccomposition of the invention is administered in an amount sufficient toelicit an effective immune response to treat a disease or arrestprogression and/or symptoms. The dosage of the adjuvants of theinvention will vary depending on the nature of the immunogen and thecondition of the subject, but should be sufficient to enhance theefficacy of the immunogen in evoking an immunogenic response. Fortherapeutic or prophylactic treatment, the amount of adjuvantadministered may range from 0.05, 0.1, 0.5, or 1 mg per kg body weight,up to about 10, 50, or 100 mg per kg body weight or more. The adjuvantsof the invention are generally non-toxic, and generally can beadministered in relatively large amount without causing life-threateningside effects.

The term “therapeutic immune response”, as used herein, refers to anincrease in humoral and/or cellular immunity, as measured by standardtechniques, which is directed toward the target antigen. Preferably, theinduced level of immunity directed toward the target antigen is at leastfour times, and preferably at least 16 times the level prior to theadministration of the immunogen. The immune response may also bemeasured qualitatively, wherein by means of a suitable in vitro or invivo assay, an arrest in progression or a remission of a neoplastic orinfectious disease in the subject is considered to indicate theinduction of a therapeutic immune response.

In the methods of the present invention, a composition comprising animmunogen and an adjuvant of the invention, combined in therapeuticallyeffective amounts, is administered to a mammal in need thereof. The term“administering” as used herein means delivering the immunogen andadjuvant of the present invention to a mammal by any method that mayachieve the result sought. They may be administered, for example,intravenously or intramuscularly. The term “mammal” as used herein isintended to include, but is not limited to, humans, laboratory animals,domestic pets and farm animals. “Therapeutically effective amount” meansan amount of the immunogen and adjuvant that, when administered to amammal, is effective in producing the desired therapeutic effect.

Compositions comprising immunogens and adjuvants of the invention may beadministered cutaneously, subcutaneously, intravenously,intramuscularly, parenterally, intrapulmonarily, intravaginally,intrarectally, nasally or topically. The composition may be delivered byinjection, orally, by aerosol, or particle bombardment.

Compositions for administration may further include various additionalmaterials, such as a pharmaceutically acceptable carrier. Suitablecarriers include any of the standard pharmaceutically accepted carriers,such as phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. Typically such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. The composition of the invention mayalso include suitable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions may be in theform of liquid or lyophilized or otherwise dried formulations and mayinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength, additives such as albumin or gelatinto prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), solubilizing agents (e.g. glycerol,polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol,parabens), bulking substances or tonicity modifiers (e.g., lactose,mannitol), covalent attachment of polymers such as polyethylene glycolto the protein, complexing with metal ions, or incorporation of thematerial into or onto particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, hydrogels, etc. or ontoliposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance.

Pharmaceutical Compositions

In certain embodiments, the present invention provides for apharmaceutical composition comprising an immunogen and an adjuvant lipidas identified herein. The immunostimulatory composition can be suitablyformulated and introduced into a subject or the environment of a cell byany means recognized for such delivery.

Such compositions typically include the agent and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in a selected solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

The compositions of the invention could also be formulated asnanoparticle formulations.

The compounds of the invention can be administered for immediate-,delayed-, modified-, sustained-, pulsed- or controlled-releaseapplications.

The pharmaceutical compositions of the invention may contain from 0.01to 99% weight-per volume of the active material.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedin a method of the invention, the therapeutically effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC50 (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of anadjuvant-containing composition of the invention targeting a disease ordisorder (i.e., an effective dosage) depends on the immunogen and targetdisease or disorder selected. For instance, single dose amounts of animmunogen of an immunogen-adjuvant composition of the inventiontargeting a disease or disorder in the range of approximately 1 pg to1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30,100, or 1000 mg may be administered. In some embodiments, 1-5 g of thecompositions can be administered.

A therapeutically effective amount of the compound of the presentinvention can be determined by methods known in the art. In addition todepending on the immunogen used, the therapeutically effectivequantities of a pharmaceutical composition of the invention will dependon the age and on the general physiological condition of the patient andthe route of administration. In certain embodiments, the therapeuticdoses will generally be between about 10 and 2000 mg/day and preferablybetween about 30 and 1500 mg/day. Other ranges may be used, including,for example, 50-500 mg/day, 50-300 mg/day and 100-200 mg/day.

Administration may be a single dose, multiple doses spaced at intervalsto allow for an immunogenic response to occur, once a day, twice a day,or more often, and may be decreased during a maintenance phase of adisease or disorder, e.g. once every second or third day instead ofevery day or twice a day. The dose and the administration frequency willdepend on the clinical signs, which confirm maintenance of the remissionphase, with the reduction or absence of at least one or more preferablymore than one clinical signs of the acute phase known to the personskilled in the art. The skilled artisan will appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of an immunogenic,adjuvant-containing composition targeting a disease, disorder orinfectious agent can include a single treatment or, optionally, caninclude a series of treatments.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. It is to be understood and expected thatvariations in the principles of invention herein disclosed may be madeby one skilled in the art and it is intended that such modifications areto be included within the scope of the present invention.

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES Example 1: Materials and Methods Mouse Strains and Cell Culture

C57BL/6J (Jax 000664), C57BL/6NJ (Jax 005304), CD14 KO (Jax 003726),caspase-1/-11 dKO mice (Jax 016621), TLR4-mutant (C3H/HeJ, Jax 000659),and wild-type control for TLR4-mutant (C3H/HeSNJ, Jax 000661) werepurchased from Jackson Labs. NLRP3 KO and ASC KO mice were kindlyprovided by Dr. T. Horng, Harvard School of Public Health. Caspase-11 KOmice were kindly provided by Dr. Junying Yuan, Harvard Medical School.Caspase-1 single KO mice were kindly provided by Thirumala-DeviKanneganti (St. Judes). DCs were differentiated from bone marrow in IMDM(Gibco), 10% B16-GM-CSF derived supernatant, 2 μM 2-mercaptoethanol and10% FBS and used after 6 day of culture. DC purity was assessed by flowcytometry and was usually higher than 90%. MO were differentiated frombone marrow in DMEM (Gibco), 30% L929 supernatant, and 10% FBS. ImmortalMΦs were cultured in DMEM supplemented with 10% L929 supernatant and 10%FBS. Splenic DCs were purified as described previously (Zanoni et al.,2012). Prior to stimulations, cultured cells were washed and re-platedin DMEM supplemented with 10% FBS at a concentration of 1×106 cells/mlin a final volume of 100 μl. For experiments using pan-caspaseinhibitor, cells were treated with zVADfmk (20 μM) 30 min beforeinflammasome activation stimuli addition. Cycloheximide (50 ng/ml) wasadded at the time of stimuli administration. Transfection with DOTAP wasperformed following the manufacturer instructions. Briefly, 375 ng ofDOTAP were added to 5 μg of LPS or 10 μg of oxPAPC in a final volume of10 μl of DMEM without FBS. 30 min later, the DOTAP/LPS or DOTAP/oxPAPCcomplex were added to the culture. FuGENE was used as previouslydescribed (Kayagaki, N. et al. (2013) Science 341, 1246-1249) totransfect LPS and oxPAPC at the indicated concentrations.

Gene Expression Analysis and ELISA

RNA was isolated from cell cultures using Qiashedder (Qiagen) andGeneJET RNA Purification Kit (Life Technologies). Purified RNA wasanalyzed for gene expression on a CFX384 real time cycler (Bio-rad)using TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems) with probespurchased from Life Technologies specific for viperin (Mm00491265_m1),IFNb1 (Mm00439552_s1), IL6 (Mm00446190_m1), caspase-1 (Mm00438023_m1),caspase-11 (Mm00432307_m1), Nlrp3 (Mm00840904_m1), Asc (Mm00445747_g1),TBP (Mm00446971_m1) or GAPDH (Mm99999915_g1). ELISA for IL-1β, IL-2,IL-17, IL-18, TNFα and IFNγ were performed using Mouse Ready-SET-GoELISA kits (eBioscience). To measure secreted cytokines, supernatantwere collected, clarified by centrifugation and stored at −20° C. Cellassociated cytokines were measured as follows: 96-well plates werecentrifuged and supernatant was discarded. 250 μl of PBS were added toeach well. Cells were frozen and thawed two times at −80° C. and thenstored at −20° C. for further analysis.

Antibodies and Reagents

E. coli LPS (Serotype O55:B5-TLRgrade™) was purchased from Enzo. OxPAPCand Pam3CSK4 were purchased from Invivogen. Oxidized PAPE-N-biotin(biotin-oxPAPC) and oxPAPC enriched in PEIPC were produced as previouslydescribed (Springstead, J. R. et al., (2012) J Lipid Res 53, 1304-1315).KOdiA-PC and DMPC were from Cayman Chemical and Avanti Polar Lipids,respectively. The following antibodies were used: HA (Roche; 3F10),MyD88 (R&D; AF3109), Actin (Sigma; A 5441), ASC (Millipore, clone2E1-7), caspase-11 (Biolegend, clone Cas11.17D9), caspase-3 (Santa Cruz,H-277), viperin (Biolegend), phospho-Stat-1 (Cell Signaling, clone58D6). The IRAK4 antibody was a gift from Shizuo Akira (OsakaUniversity). For flow cytometry based assays, the fluorophore-conjugatedantibodies were used as the following: PE anti-TLR4 (Biolegend; cloneSa15-21), PE/Cy7 anti-TLR4/MD2 (Biolegend; clone MTS510), FITC anti-CD14(eBioscience; clone Sa2-8), APC anti-CD14 (ebioscience; clone Sa-28).PE-anti-MHC class II and APC anti-CD40 antibodies were from eBioscience.Annexin V and 7-AAD viability stain solution was purchased fromBioLegend. Incomplete Freund's Adjuvant (F5506) and cycloheximide(C1988) were purchased from Sigma. DOTAP was purchased from Roche.FuGENE 2000 was from Promega. Endotoxin-free OVA was purchased fromHyglos/Biovendor. Recombinant IFNβ was from R&D Systems. Pierce LDHCytotoxicity Assay Kit was purchased from Life Technologies.

Protein Purification and In Vitro Protein-Lipid Interactions

For studies to measure the direct binding of oxPAPC to caspase-11,proteins and SPR analysis was performed as described (Shi, J., et al.,(2014b) Nature). Briefly, the recombinant catalytic mutant caspase-11full length (C254A) and caspase-11 ΔN59 (C254A) were purified from P3baculovirus infected SF-21 insect cells that were cultured in Sf900™ IISFM for 72 h at 28° C. Cells were lysed in lysis buffer containing 1%Triton X-100, 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 50 mM imidazole and5 mM 2-mercaptoethanol. Ni-NTA beads (Qiagen) were used to purifyHis-tagged proteins from lysates. Proteins were released from the beadswith elution buffer containing 50 mM Tris-HCl (pH 7.6), 250 mM imidazoleand 300 mM NaCl. Imidazole was removed by dialysis. The proteins werefurther purified with HiTrap Q column and Superdex G200 column (GEHealthcare Life Sciences) to reach high homogeneity.

For surface plasmon resonance (SPR) analysis, BIAcore T100 SPRinstrument (GE Healthcare) was used to measure the ligand bindingkinetics. The assays were performed in a buffer containing 150 mM NaCl,3 mM EDTA, 50 mM HEPES (pH 7.5) and 0.005% Tween-20 at 25° C. CMS sensorchip was first activated with 1:1 mixed 0.1 MN-ethyl-N′-(3-diethylaminopropyl)-carbodiimide and 0.1 MN-hydroxysuccinimide solution at a flow rate of 10 μL/min for 7 min.Catalytic mutant caspase-11 full length (C254A) and caspase-11 ΔN59(C254A) were diluted to a concentration of 20 μg/mL with 10 mM sodiumacetate (pH 5.0) and immobilized to about 3100 response units and 3300response units respectively. 10 mM sodium acetate diluted rabbit IgGprotein (10 μg/ml) was immobilized to 3400 response units and treated asnegative control. 1 M ethanolamine (pH 8.5) was flowed over the CMS chipto block all the remaining protein binding sites for 7 min (flow rate 10μL/min). The ligands were passed over the flow cell and adjacent controlflow cell (activated and blocked as targeting flow cells, but no proteinwas immobilized) for 1 min at a flow rate of 30 μL/min. A dissociationprocess was performed for 2 min at a flow rate of 30 μL/min. The boundligands were removed with 20 mM NaOH washed for 20 seconds. The KDvalues were calculated by fitting result curves (subtracted the controlflow cell value) to a 1:1 Langmuir binding model with the BIAcore T100evaluation software.

For caspase-11 oligomerization and enzymatic activity assays, fulllength mouse caspase 11 was cloned into pFastBac™HT A vector(Invitrogen) with a TEV cleavable N-terminal 6×His tag using EcoRI andXhoI restriction sites. The protein was expressed using the Bac-to-Bacbaculovirus-insect cell system. After 48 h-infection, the Sf9 cells thatexpressed His-caspase11 protein were harvested by centrifugation at2,000 rpm for 20 min. The cell pellets were resuspended in a lysisbuffer containing 20 mM HEPES at pH 7.5, 150 mM NaCl, 5 mMtris(2-carboxyethyl)phosphine (TCEP), 20 mM imidazole and a proteaseinhibitor cocktail, and homogenized by ultrasonication. The cell lysatewas clarified by ultracentrifugation at 42,000 rpm at 4° C. for 2 hours.The supernatant containing the target protein was incubated with Ni-NTAresin (Qiagen) that was pre-equilibrated with the lysis buffer for 1hour at 4° C. After incubation, the resin-supernatant mixture was pouredinto a column and the resin was washed with the lysis buffer. Theproteins were eluted by the lysis buffer supplemented with 500 mMimidazole, and further purified by size exclusion chromatography.

To measure the ability of oxPAPC to oligomerize caspase-11, monomer oroligomer fractions of His-caspase11 were incubated with oxPAPC for 2hours on ice, and then analyzed with Superdex 200 (10/300).

To characterize the association between biotinylated oxPAPC andHA-tagged caspase-11 in cell lysates, 293T cells were transientlytransfected with pcDNA vector expressing indicated caspase-11 alleles(WT, K19E and 3K (K62E K63E K64E)) with the C-terminal fusion of an HAepitope. 48 hours after transfection, cells were collected with cold PBSand lysed in 1 ml lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150mM NaCl, 10% Glycerol and 1% NP-40 supplemented with complete proteaseinhibitor (Roche). Cells were lysed on ice for 30 min and the whole cellextracts were collected into a new tube after spinning at 14000×g for 15minutes in a table-top centrifuge in the cold room. For each of thewhole cell lysate containing the indicated caspase 11 alleles, 100 μLwas taken out and saved as input. The remaining 900 μl lysates wereequally split into 3 tubes with 300 μL each tube. 1 μg of biotinylatedLPS and 10 μg of the biotinylated oxPAPC were added to the first and thesecond tube, respectively. The third tube was left as a mock control (tomonitor the extent of non-specific binding between indicated caspase-11alleles to the streptavidin beads). The biotinylated ligands and lysateswere mixed and incubated at 4° C. on a nutator for either 6 hours orovernight. To capture the ligand-caspase-11 complex, streptavidin beads(20 μL bed volume) were then added to all of the tubes (including themock control that was never treated with any biotinylated ligands). Thiscapturing step was allowed to proceed at 4° C. for another 2 to 3 hours.The beads were then washed with the lysis buffer for 3 times and finally50 μL of SDS loading buffer was added. The protein complexes werefurther eluted by heating at 65° C. for 15 minutes. 25 μL of the elutedprotein complexes were separated by SDS-PAGE and the proteins retainedby biotinylated ligands were detected by western analysis.

Capture of Endogenous Caspases Through Protein-Lipid Interactions

S100 fractions from iMΦs were prepared as follows. Confluent iBMDMscultured in complete DMEM medium were harvested with ice-cold PBS plusEDTA (0.4 mM). Cells were then washed once with homogenization buffer(HB) (20 mM HEPES/KOH, pH7.9, 250 mM sucrose, 0.5 mM EGTA) supplementedwith complete protease inhibitor tablets (Roche). Cells were lysedmechanically by subjecting to 20 strokes of douncing in the Wheaton™Dounce Dura-Grind™ Tissue Grinder. The extent of cell lysis wasmonitored by Trypan blue staining to ensure that more than 80% of thecells were lysed. The crude lysates were then spun at 800×g for 10 minat 4° C. to remove unbroken cells and nuclear components. Thepost-nucleus supernatant were collected and spun at 13,000×g for 10 minat 4° C. to remove large organelles and membranes. Finally, the clearedlysates were transferred to Beckman polycarbonate ultracentrifugationtubes (343778) and spun at 100,000×g for 1 hour at 4° C. to removeresidual membrane components. The resultant 5100 supernatant (containingsoluble cytosolic proteins) were stored at −80° C. with a proteinconcentration at 2 mg/mL or used as a source of endogenous caspases tobe captured by biotinylated lipids. 1 mg of 5100 supernatant wasincubated with 15 μg of biotin-oxPAPC for 12-16 hours at 4° C. on anutator. Streptavidin agarose resin (Pierre, P. et al., (1997) Nature388, 787-792) was used to capture the endogenous protein complexesassociated with Biotinylated oxPAPC (with 20 μL bed volume resin perreaction) for 1 to 2 hours at 4° C. on a nutator. The protein complexescaptured by the resin were then washed for 4 times withdetergent-containing washing buffer (50 mM Tris-HCl, pH 7.5, 150 mMNaCl, 10% Glycerol, 1% NP-40) and further eluted by incubated with 60 μLof SDS loading buffer at 65° C. for 20 min. One third of the eluate wasseparated by SDS-PAGE and endogenous caspases retained by biotin-oxPAPCwere detected by Western blotting using designated antibodies.

Caspase-11 Activity Assay

5 μM His-Caspase-11 with or without lipid (LPS, oxPAPC and DMPC) wereused for caspase activity assay in the reaction buffer containing 50 mMHEPES (pH 7.5); 10% (v/v) glycerol; 10 mM DTT; 1.0 mM EDTA; 0.2% (w/v)BSA in a Corning® 96 Well Half Area Black Flat Bottom Microplate.Reactions were started by adding substrate YEVD-AMC at a finalconcentration of 10 μM. Data were collected with the SpectraMax M5eMulti-Mode Microplate Reader (Molecular Devices) using excitation at 385nm and emission at 460 nm with an auto-cutoff filter at 455 nm.

Flow Cytometry

primary bone marrow derived MΦs and DCs or splenic DCs (0.5×10⁶) of theindicated genotypes were treated with E. coli LPS, oxPAPC or chemicalinhibitors for the indicated time points at 37° C. Cells were thenwashed with 1 mL cold PBS and stained for appropriate antibodies on icefor 20 to 30 minutes. 2% mouse serum or rat serum were used as theblocking reagent to reduce non-specific binding of the antibodies. Thestained cells were then washed with 1 mL cold PBS and resuspended in 200μL PBS. Staining of the surface receptors was analyzed with BD FACSCantoII. The mean fluorescence intensity (MFI) of CD14, TLR4 fromunstimulated or stimulated cells was recorded. The percentage of surfacereceptor staining at indicated time points, which is the ratio of theMFI values measured from the stimulated cells to those measured from theunstimulated cells, was plotted to reflect the efficiency of receptorendocytosis. For measuring the extent of TLR4/MD-2 dimerization, thepercentage of TLR4/MD2 dimer was calculated by 100%−the percentage ofTLR4/MD-2 monomer. The percentage of the TLR4/MD-2 monomer wasdetermined by the ratio of the MFI values (obtained from MTS510 antibodystaining) of the stimulated cells to those of the unstimulated cellswere indicated. Cells were stained with anti-MHC class II or anti-CD40antibodies.

Western Blotting and Myddosome Formation

For Western blotting, iMΦs (5×10⁶) were stimulated with ligands forindicated periods, and subsequently lysed in 700 μL of lysis buffercontaining 1% NP-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl. Proteaseinhibitors and phosphatase inhibitors were added just prior to celllysis. Immunoblotting was performed using standard molecular biologytechniques.

For myddosome formation, (3×10⁶) were stimulated with ligands forindicated periods, and subsequently lysed in 700 μL of lysis buffercontaining 1% NP-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl. Proteaseinhibitors and phosphatase inhibitors were added just prior to celllysis. Lysates were spun at top speed for 15 minutes at a table-topcentrifuge in the cold room (at 4° C.). The cleared supernatants werecollected and 80 μL of the supernatant was saved as total extract. 1 μgof the anti-MyD88 antibody and 15 μL (bed volume) of protein G Sepharosewere added to the remaining supernatants and the incubation were allowedto proceed for overnight at 4° C. on a nutator. The beads were thenwashed for 3 times with lysis buffer, and 60 μL of SDS loading bufferwas added. The protein complexes were further eluted by heating at 65°C. for 15 minutes. A portion of eluted protein complexes (20 μL) wereseparated by SDS-PAGE and visualized by western blotting using indicatedantibodies.

Immunofluorescence

BMDCs cells were treated with LPS (1 μg/mL) for 3 hours beforeinflammasome-inducing stimuli challenge. For live or permeabilityexperiment, MitotTacker CMX-ROS (Life Technologies) or Zombie Red(BioLegend) dyes were used in according to manufacturer's instructionsbefore fixation with 4% paraformaldehyde. After permeabilization stepusing 0.1% Triton X-100 0.2% BSA-PBS, cells were blocked in 2% BSA-PBSand incubated with rabbit anti-ASC pAb (AL177, Adipogen) and mouseanti-caspase-1 mAb (Casper-1, Adipogen) followed by Alexa Fluor488-conjugated chicken anti-rabbit IgG (Life Technologies) and AlexaFluor 568-conjugated goat anti-mouse IgG (Life Technologies) diluted inblocking buffer. Nuclei were counterstained with DAPI (LifeTechnologies) or DRAQ5 (BioLegend). A Zeiss Axiovert 200M confocalmicroscope or an Olympus BX41 fluorescence microscope was used toacquire images.

PI Permeabilization Assay

BMDCs or BMMs were seeded into black 96-well tissue culture plates withtransparent bottom and treated with priming stimuli for 3 h. Aftergently wash with PBS, 100 μl of pre-warmed staining solution (5 μM PI,5% FBS, 20 mM HEPES, no phenol red containing MgCl₂ and CaCl₂ HBSS) wasadded to each well and incubated for 5 minutes at 37° C. 5% CO₂.Immediately before reading, 100 μL staining solution without PIcontaining 2× inflammasome-inducing stimuli was added into appropriatewells. 0.1% Triton X-100 was used as positive control for the maximumpermeability. The increasing fluorescence intensity was recorded for 3hours at 37° C. in continuum using a FLUOstar Omega microplate reader(BMG labtech) with 544 nm excitation and 620-10 nm emission filters.

In Vivo Immunization and In Vitro Re-Stimulation

WT C57BL/6NJ and Caspase-1/-11 dKO C57BL/6NJ mice were immunized on theupper back (one injection over each shoulder) with either 150 μg/mouseendotoxin-free OVA plus 7 μg/mouse LPS emulsified in incomplete Freund'sadjuvant or with 150 μg/mouse endotoxin-free OVA, plus 65 μg/mouseoxPAPC, plus 7 μg/mouse LPS emulsified in incomplete Freund's adjuvant.CD4+ T cells were isolated from the draining lymph nodes 7 or 40 daysafter immunization by magnetic cell sorting with anti-CD4 beads(Miltenyi Biotech). The cells were seeded in 96-well plates at aconcentration of 100,000 cells per well in the presence of 100,000 DCand of serial dilutions of OVA, starting at 1 mg/ml. Secretion of IFNγ,IL-17 and IL-2 were measured by ELISA 5 days later.

HSV Infections Assay of Viral Replication

Mice were housed in accordance with institutional and NIH guidelines oncare and use of animals in research, and all procedures were approved bythe Institutional Animal Care and Use Committee of Harvard MedicalSchool. The indicated mouse strains were anesthetized in an isofluranechamber followed by intraperitoneal injections of ketamine (3.7mg/mouse) and xylazine hydrochloride (0.5 mg/mouse). The corneas werescarified, and infections were carried as described previously (Cliffe,A. R. et al., (2009) Journal of virology 83, 8182-8190). To measureviral replication in the eye, swabs of tear film were collected usingsterile polyester applicators (Puritan) for the first 5 dpi, and virusin the tear films from the eye was titrated on Vero cells as describedpreviously (Coen, D. M. et al., (1989) Proc Natl Acad Sci USA 86,47364740).

Statistical Analysis

Hypotheses were tested with two-tail t-tests in single pairwisecomparisons. p-values, calculated with Excel (Microsoft) are coded byasterisks: <0.05 (*), <0.01 (**), <0.001 (***).

Example 2. Identification of oxPAPC as Both a TLR4 Antagonist and a CD14Agonist

Oxidized phospholipids such as oxPAPC have a confusing history, havingbeen reported to act as either activators or inhibitors of inflammation.Some studies have demonstrated that oxPAPC can inhibit TLR4-dependentinflammatory cytokine expression induced by LPS in a concentrationdependent manner (Bochkov, V. N. et al., (2002) Nature 419, 77-81;Erridge, C. et al., (2008) The Journal of biological chemistry 283,24748-24759; Oskolkova, O. V. et al. (2010) J Immunol 185, 7706-7712),whereas others have reported oxPAPC to be an activator of TLR4-dependentinflammatory responses (Imai, Y. et al. (2008) Cell 133, 235-249;Shirey, K. A. et al. (2013) Nature 497, 498-502).

To determine the activities of oxPAPC and PAPC, the abilities of theselipids to engage the LPS receptors TLR4 and CD14 was examined inimmortal murine bone marrow derived macrophages (BMDM; alternativelyiMϕ) from mice. Side-by-side comparisons of LPS- and oxPAPC-stimulatedcells were performed to assess the potential of these molecules toinduce the expression of known TLR4-dependent genes (FIGS. 1 and 2 ). Ascompared to LPS, which induced the robust expression of the cytokinesIL-1β and interferon beta (IFNβ) (FIG. 2 ), and the IFN-stimulated geneviperin, oxPAPC was unable to upregulate these genes (FIG. 43B). It waspossible that other TLR4-dependent genes than those assayed were beingactivated by oxPAPC.

Several concentrations of oxPAPC were assessed in these studies, all ofwhich were similar to those reported to be present in inflamed ordamaged tissues in vivo (Oskolkova, O. V. et al. (2010) J Immunol 185,7706-7712). TLR4 dimerization was assessed by flow cytometry, using anantibody that only detects TLR4 monomers. Dimerization was identified tobe induced by LPS, but not by oxPAPC treatment (FIG. 43A). To complementthese analyses, the inducible interactions between the receptor-proximalproteins MyD88 and IRAK4 were also examined.

These proteins form a supramolecular organizing center (SMOC) called themyddosome (Kagan, J. C. et al., (2014) Nat Rev Immunol 14, 821-826; Lin,S. C. et al., (2010) Nature 465, 885-890; Motshwene, P. G. et al.,(2009) J Biol Chem 284, 25404-25411), which wis only assembled inresponse to TLR activation (Bonham et al., 2014). Thus, detection of amyddosome can be used as a general readout of TLR activation. WhereasLPS induced the formation of a MyD88-IRAK4-containing myddosome within30 minutes of treatment, oxPAPC was unable to elicit any detectableassociation between these proteins (FIGS. 43C, and 49A). Furthermore,oxPAPC-treated cells contained no detectable amounts of phosphorylatedSTAT1 or the IFN-stimulated gene viperin (FIG. 1 , FIG. 43D, and FIG.49B), both of which were abundant upon LPS treatment. These dataindicated that oxPAPC was not a mimic of LPS, and had little or noability to directly activate TLR4 directly in BMDM.

In cell-free or overexpression systems, oxPAPC acted as an inhibitor ofTLR4 signaling events by competing with LPS for access to either CD14 orthe LPS-binding protein MD-2 (Bochkov, V. N. et al., (2002) Nature 419,77-81; Erridge, C. et al., (2008) The Journal of Biological Chemistry283, 24748-24759), the latter of which was responsible for crosslinkingand activating TLR4. However, the ability of oxPAPC to engage the TLR4regulators has mainly been examined in cell-free systems or epithelialcells. The degree of inhibition by oxPAPC was affected by altering theratio of LPS and oxPAPC administration (FIGS. 1-5 ), which indicatedthat these two factors were likely competing for the same binding sitewith CD14. Consistent with such competition over a single binding site,a mutant CD14 allele that could not bind LPS was not endocytosed even inthe presence of oxPAPC or LPS.

To determine if oxPAPC engaged CD14 in or MD-2 in BMDM, several assayswere used to monitor the inducible dimerization or endocytosis ofcandidate receptors by flow cytometry. As reported previously, LPStreatment caused the endocytosis of CD14 and TLR4, which resulted in theloss of surface staining for these proteins (Zanoni, I. et al., (2011)Cell 147, 868-880). Interestingly, oxPAPC was unable to induce theendocytosis of TLR4 (FIG. 4 ), but was able to promote rapid endocytosisof CD14 (FIG. 3 , FIG. 44A, and FIG. 50A). This rapid internalization ofCD14 induced by oxPAPC therefore created a CD14-deficiency at the cellsurface. Without wishing to be bound by theory, it is believed that thisdeficiency of CD14 at the cell surface could explain the ability of thislipid to block TLR4 signaling. Indeed, oxPAPC treated cells that weresubsequently treated with LPS exhibited defects in TLR4 endocytosis andTLR4-induced gene expression.

CD14 surface abundance resulted from the antagonistic actions of CD14endocytosis and resynthesis (Tan, Y. et al., (2015). Immunity 43,909-922), and was most clearly observed under conditions that preventthe latter. Consequently, the extent of oxPAPC- or LPS-induced CD14endocytosis was enhanced under conditions where protein synthesis wasblocked with cycloheximide (FIG. 50B). Cycloheximide treatment did notaffect either TLR4 internalization or dimerization (FIGS. 50C and 50D).Primary bone marrow derived MΦ and bone marrow derived DCs behavedsimilarly to in that oxPAPC promoted CD14 endocytosis, but not TLR4dimerization or endocytosis (FIG. 44B). The endocytosis of CD14 (but notTLR4) induced by oxPAPC therefore created a CD14-deficiency at the cellsurface, which likely explains the ability of this lipid to block TLR4signaling. Indeed, oxPAPC-treated cells that were subsequently treatedwith LPS exhibited defects in TLR4 dimerization, endocytosis, TNFαsecretion and STAT1 phosphorylation, with the latter two being classicreadouts of TLR4 signaling (FIGS. 44A and 44F).

To address the possibility that CD14 used a similar mechanism tointeract with a PAMP (LPS) and a DAMP (oxPAPC), the amino acids withinCD14 required for interactions with these lipids were examined. TheLPS-binding domain of CD14 was previously identified to be a largehydrophobic pocket that is comprised of four distinct regions of theprimary amino acid sequence (Kim, J. I. et al., (2005) J Biol Chem 280,1134711351). CD14 alleles that contained mutations in either one region(1R) or two regions (2R) retained the ability to form a complex withbiotinylated LPS, whereas mutations of all four regions (4R) in CD14abolished LPS-binding activity (Tan, Y. et al., (2015). Immunity 43,909-922). Each of these mutant CD14 alleles encoded full-length foldedproteins that were transported to the cell surface (Tan, Y. et al.,(2015). Immunity 43, 909-922). Notably, the 4R mutant was also defectivefor interactions with biotinylated oxPAPC (FIG. 44E). Furthermore, whenstably introduced into CD14 knockout (KO) iMΦ, the 4R mutant CD14 wasnot internalized in response to LPS or oxPAPC treatments (FIG. 44F).These data therefore indicated the same amino acids within CD14 promotedinteractions with a DAMP (oxPAPC) and a PAMP (LPS), and providedmolecular support for the conclusion that oxPAPC can be considered aselective LPS mimic (i.e. in the case of CD14-dependent activities).Overall, these data indicated that oxPAPC was not an activator of TLR4,but was an activator of CD14. This ability to dissociate CD14 and TLR4endocytosis likely explains how oxPAPC functions as a TLR4 antagonist.

Example 3. oxPAPC Promoted the Activation of the NLRP3 Inflammasome inDendritic Cells (DCs)

While the above example indicated that oxPAPC was not an activator ofinflammation, some studies have demonstrated a pro-inflammatory functionof these lipids (Imai, Y. et al. (2008) Cell 133, 235-249; Shirey, K. A.et al. (2013) Nature 497, 498-502). It was considered that some DAMPscould not elicit pro-inflammatory responses from naïve cells, but thatthey could induce cytokine release from cells previously exposed tomicrobial products. For example, extracellular ATP was described to haveactivated inflammasome-dependent release of IL-1β from cells that hadbeen primed with TLR ligands (Petrilli, V. et al., (2007) Currentopinion in immunology 19, 615-622).

As shown in FIGS. 6 and 7 , PAPCs, including oxPAPC component lipidKOdiA-PC (1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine),activated the inflammasome in DCs.

As shown in FIG. 8 , CD14 regulated inflammasome activation in responseto PAPCs. Inflammasome activation in response to PAPCs was CD14specific, although PAPCs could also induce CD36 internalization (FIG. 9). CD14 regulated PAPCs-mediated inflammasome activation independentlyof Type I IFNs (FIG. 10 ).

Regulation of Caspase-1 and Caspase-11 expression was similar in wt DCsand Cd14−/−DCs (FIG. 11 ). PAPCs induced inflammasome activation in acell type specific manner (FIG. 12 ).

It was also identified that other PAMPs primed PAPCs-inducedinflammasome activation (FIGS. 13 and 14 ).

Notably, not all modified PCs induced inflammasome activation (FIG. 15).

To determine whether oxPAPC had pro-inflammatory functions in acontext-dependent way, the release of IL-1β from primary BMDM or BMDCthat were pretreated (or not) with LPS was examined. Consistent withprevious observations (Petrilli, V. et al., (2007) Current opinion inimmunology 19, 615-622), LPS-pretreatment enabled ATP to elicit IL-1βrelease from DCs in a dose-dependent manner (FIG. 51A). Remarkably,oxPAPC had similar activities, but in a cell-type dependent manner.Interestingly, oxPAPC was also able to induce IL-1β secretion, but onlyin LPS-primed DCs (FIG. 45A). oxPAPC did not induce IL-1β release fromnaïve cells, but LPS pretreatment of DCs enabled oxPAPC to promote IL-1βrelease in a dose-dependent manner (FIG. 45A, and FIG. 51B).

Without wishing to be bound by theory, IL-1β release is typicallymediated by inflammasomes, which are cytoplasmic complexes of proteinsthat trigger the processing and atypical secretion of IL-1 familymembers (Petrilli, V. et al., (2007) Current opinion in immunology 19,615-622).

To determine whether the above-identified oxPAPC-mediated release ofIL-1β was an inflammasome-dependent event, the activities of this lipidwere examined in BMDCs derived from either caspase-1/caspase-11 doubleknockout (KO) mice, or mice lacking ASC (also known as Pycard), whichwas a common adaptor protein involved in inflammasome assembly(Martinon, F. et al., (2002) Molecular cell 10, 417-426). oxPAPC (orATP) mediated release of IL-1β was completely lost from BMDCs lackingcaspase-1/-11 (FIG. 18 ) or ASC (FIG. 17 , and FIGS. 45B-C), anobservation that provided definitive genetic proof for the requirementof inflammasome(s) in oxPAPC-induced cellular responses. Noting thatNLRP3 was among the most common upstream activators of inflammasomes(Ye, Z., and Ting, J. P. (2008) Current opinion in immunology 20, 3-9),oxPAPC-mediated IL-1β release was also examined in NLRP3-deficientBMDCs. oxPAPC-mediated IL-1β release was thus identified as anNLRP3-dependent process, as oxPAPC was unable to induce IL-1β releasefrom NLRP3-deficient BMDCs (FIG. 16 ), and from NLRP3 KO DCs (FIG. 45D).ATP-mediated IL-1β was also NLRP3 dependent, as expected. Importantly,no inflammasome regulator was required for TNFα secretion (FIGS.45B-45D), which indicated that TLR4-induced gene expression occurredindependent of inflammasome activation.

Commercially available (and natural) oxPAPC contains a mixture ofdifferent oxidized species. To determine whether alternative sources ofoxPAPC yielded similar activities, a custom-made oxPAPC identified to beenriched in PEIPC (1-palmitoyl-2-(5,6epoxyisoprostanoyl)-sn-glycero-3-phosphocholine), the most activecomponent of oxPAPC (Springstead et al., 2012), was used. Side-by-sideanalysis of the two different oxPAPCs yielded similar results (FIG.45A), confirming that independent of the source, oxPAPC induced IL-1βrelease in LPS-primed DCs. In contrast to the effects observed for IL-1βrelease, cell-associated IL-1β levels were similar when comparing cellsstimulated with LPS alone, LPS/oxPAPC or LPS/ATP (FIGS. 45A, 51B, and51C). This latter observation was consistent with the finding thatoxPAPC could only act as an inhibitor of TLR4 signaling if cells werepretreated with this DAMP.

To determine the specificity of the effects of oxPAPC oninflammasome-mediated events (e.g., IL-1β release), the effect of thislipid upon the release of a classic TLR-dependent cytokine, TNFα wasexamined. oxPAPC neither promoted nor inhibited the release of TNFα fromDCs (FIG. 51D). Additionally, when DCs were co-treated at the same timewith LPS/ATP or LPS/oxPAPC (i.e. no priming), IL-1β was released only byoxPAPC-treated DCs (FIG. 51E), which indicated differences in thecapacity of these two DAMPs to regulate IL-1β secretion. When adifferent phosphocholine variant,1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was used, it wasunable to elicit IL-1β release (FIG. 51F). In contrast, a purifiedcomponent of oxPAPC, 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl)phosphatidylcholine, or KOdiA-PC, was able to elicit IL-1β secretion(FIG. 51F). In all cases, TNFα secretion was not affected byphosphocholine treatment (FIG. 51F). These data established the specificability of oxPAPC to promote IL-1β release, without affecting TLR4signal transduction in LPS-primed DCs.

oxPAPC-induced inflammasome activation, but not ATP-induced inflammasomeactivation, was Caspase-11 dependent (FIG. 19 ). Indeed, PAPCs-inducedinflammasome activation after priming with both LPS and Pam3 requiredCaspase-11 (FIG. 20 ). Thus, biotinylated forms of PAPCs werecontemplated as a tool for study of Caspase-11 activation.

Biotinylated PAPCs were identified to potently induce CD14internalization (FIG. 21 ), yet did not induce TLR4 internalization(FIG. 22 ) nor IL-1β secretion (FIG. 23 ). In vitro binding assays forbiotinylated LPS, OxPac and Pac to Caspase 11 and MD-2 appeared toidentify complex formation in a caspase 11-dependent manner (FIG. 24 ).Indeed, in a Bio-LPS pull-down assay, PAPC acted as a dose-dependentcompetitor (FIG. 25 ). In such assays, Biotin-LPS was used at 5 μg perpull-down assay, while PAPC was used at 5, 50 and 500 μg to compete withLPS binding to MD-2 and Caspase-11, respectively. The competition waseffective at the ratio of 1:100 (LPS:PAPC).

oxPAPC and LPS likely bound the same domain of CD14 (FIG. 26 ). However,while LPS treatment affected DC survival (FIG. 27 ), PAPCs treatment ofprimed DCs favored DC survival (FIG. 28 ). This pro-survival effect ofPAPCs was not CD14-dependent (FIG. 29 ). Inflammasome activation wastypically associated with the release of IL-1β and the subsequent deathof the activated cell. The absence of oxPAPC killing of BMDCs was apositive result further favoring use of oxPAPC as an adjuvant.

As shown in FIG. 30 , P2C and P3C alone supported DC survival. P2C andP3C primed DC did not increase their survival in response to PAPCstreatment (FIG. 31 ).

Inflammasome was efficiently activated by co-administration of primingstimuli and PAPCs but not ATP (FIG. 32 ). Co-administration of primingstimuli and PAPCs did not alter NF-κB activation in wild-type DCs (FIG.33 ).

In the absence of CD14, oxPAPC acted as an antagonist of TLR4 signaling(FIG. 34 ).

In view of the observed CD14-dependence of the oxPAPC effect, it waspossible that CD14 acted as a chaperone to clear (“clean”) PAPCs fromthe extracellular space.

Co-administration of LPS and oxPAPC affected TLR4 internalization (FIG.35 ), CD14 internalization (FIG. 36 ) and partially affected TLR4dimerization (FIG. 37 ).

To summarize certain of the above results:

(1) Specific modified PCs (PAPCs but not DMPC) induced inflammasomeactivation;

(2) PAPCs-dependent but not ATP-dependent inflammasome activation wascell type specific (DCs but not Macs);

(3) Inflammasome activation via PAPCs but not ATP required CD14;

(4) Inflammasome activation via PAPCs but not ATP required Caspase-11;

(5) ATP but not PAPCs induced pyroptosis in DCs;

(6) PAPCs favored DC survival in a CD14-independent way; and

(7) PAPCs, but not ATP, were identified to induce inflammasomeactivation when co-administered with the priming stimuli.

Thus, PAPCs were identified as a potent, natural adjuvant that couldincrease adaptive immune responses.

LPS and Rhodo LPS were also assessed for inflammasome activation. Asshown in FIG. 38 , Rhodo LPS was a potent inducer of inflammasomeactivation. LPS-induced inflammasome activation was Nlrp3-dependent(FIG. 39 ), Asc-dependent (FIG. 40 ), and Casp1/11-dependent (FIG. 41 ).Meanwhile, Rhodo LPS-induced inflammasome activation wasCD14-independent (FIG. 42 ). Thus, Rhodo LPS was also identified as anon-canonical inflammasome-activating lipid, though the effect appearedto be independent from CD14 (thereby distinguishing the apparentmechanism for Rhodo LPS from that seen for oxPAPCs).

Example 4: oxPAPC Did not Promote IL-1R Release from Macrophages

All well-defined inflammasome activators tested promoted IL-1β releasefrom MΦs. To examine whether oxPAPC possessed this ability, experimentssimilar to those described above were performed upon primary bonemarrow-derived MΦs. Interestingly, oxPAPC was unable to elicit IL-1βrelease under any condition examined in MΦs (FIG. 45E), whereas ATPpromoted the efficient release of IL-1β from these cells in adose-dependent manner (FIG. 51A). These data identified oxPAPC as a celltype-specific activator of inflammasome activities.

To better understand how DCs uniquely respond to oxPAPC, the response tothe priming stage of inflammasome activation was evaluated. Whenexamined side-by-side, DCs produced more TNFα than MΦs produced inresponse to LPS (FIG. 51D). These results indicated that DCs were better“primed” than MΦs. However, IFNγ-treated MΦs were primed as well as DCs,yet they still did not release IL-1β in response to oxPAPC (FIG. 51G).The differential responsiveness of DCs and MΦs to oxPAPC was likelymanifested after the priming step had occurred, at the stage ofinflammasome activation.

It was contemplated that a unique factor in DCs might exist thattransports oxPAPC to the cytosol, where oxPAPC then activatesinflammasome-mediated IL-1β release. This possibility was examined bytransfecting oxPAPC into the MΦ cytosol directly. While this methodpromoted IL-1β release from DCs that were primed with the TLR2 ligandPam3CSK, primed MΦs were still unable to induce such a response (FIG.45F). LPS transfection in the cytoplasm was used as a positive control(FIG. 45F) (Hagar, J. A. et al., (2013) Science 341, 12501253; Kayagaki,N. et al. (2013) Science 341, 1246-1249). These findings indicated thatsome factor(s) does exist in the cytosol of MΦs (or DCs) that allowsoxPAPC to activate the latter.

To understand inflammasome activities present in MΦs and DCs moregenerally, another inflammasome activator, ATP, which promoted IL-1βrelease from both types of cells (FIG. 51A) was examined. Interestingly,DCs and MΦs died in response to LPS+ATP treatments with similarkinetics, but while these cells released very different amounts of IL-1β(FIG. 45G) and expressed very different levels of ASC (FIGS. 51H-51I),none of the other components of the canonical and non-canonicalinflammasome (FIG. 51I) were released or expressed. In MΦs, there was aperfect correlation between the extent of cell death and the extent ofIL-1β release, an observation consistent with dying cells releasing thiscytokine (FIG. 45G). In contrast, maximal amounts of IL-1β were releasedfrom DCs when minimal death was observed, an observation that wasconsistent with living cells releasing this cytokine (FIG. 45G).Collectively, these data highlighted fundamental differences in theactivities of inflammasomes in MΦs and DCs, and indicated that oxPAPCwas an activator of inflammasomes, specifically in the DCs.

Example 5: oxPAPC Promoted IL-1β Release Via Non-Canonical Inflammasome,Independent of TLR4

Caspase-11 is a known protease that binds to cytosolic LPS and promotesthe assembly of non-canonical inflammasomes and the release of IL-1β(Hagar, J. A. et al., (2013) Science 341, 12501253; Kayagaki, N. et al.(2013) Science 341, 1246-1249; Shi, J. et al., (2014a) Nature 514,187-192). Since oxPAPC can mimic LPS and activate CD14 endocytosis,oxPAPC was also evaluated for its ability to activate caspase-11dependent responses. Remarkably, oxPAPC-mediated IL-1β release waslargely abolished in caspase-11 KO DCs (FIG. 46A). As expected,ATP-mediated IL-1β release remained intact in caspase-11 KO cells (FIG.46A). In all cases, TNFα secretion was not affected (FIG. 46B). Thisdistinction between oxPAPC and ATP, in terms of caspase-11 dependentIL-1β release, eliminated the possibility that the activities of oxPAPCwere mediated by the indirect release of ATP from cells.

To complement these functional analyses, microscopic examination ofindividual DCs revealed that both oxPAPC and ATP induced the formationof ASC and caspase-1-containing “specks” in LPS-pretreated DCs (FIG.46C). Of note, these experiments were performed using doses of ATP (1mM) and oxPAPC (120 μM) that induced the release of similar levels ofIL-1β (FIG. 51C). Although the kinetics of speck formation in responseto oxPAPC was delayed compared to ATP, specks were formed in a similaramount of cells (FIGS. 52A-52B)). These structures were only formedunder conditions where IL-1β was released, and were recognized asindividual inflammasomes (Stutz, A. et al., (2013) Methods in molecularbiology 1040, 91-101). Interestingly, caspase-11 was required for theformation of ASC/caspase-1 containing specks in response to oxPAPC, butnot ATP (FIGS. 46C and 52B). Without wishing to be bound by theory,Caspase-11 was likely required for oxPAPC-induced IL-1β release becausethis protein was required for non-inflammasome assembly.

Consistent with the idea that oxPAPC did not require TLR4 to exert itsfunctions, the ability of oxPAPC to activate IL-1β release was notdependent upon TLR4 signaling. Indeed, cells primed with the TLR2ligand, Pam3CSK, or the TLR9 ligand, CpG, elicited similar responses asthose primed with LPS (FIGS. 52C-52D). As was observed for LPS-primedcells, IL-1β release from Pam3CSK-primed DCs required NLRP3, ASC andcaspase-11 (FIG. 52C). ATP-mediated IL-1β release after Pam3CSK primingremained intact in caspase-11 KO cells, but not caspase-1/-11 dKO cells(FIG. 52C). All genotypes of DCs permitted comparable levels of TNFαsecretion (FIG. 52C). To further dissociate any possible activity ofoxPAPC on TLR4, C3H/HeJ DCs (which were naturally unresponsive to LPSdue to a mutation in TLR4 TIR domain) (Poltorak, A. et al. (1998)Science 282, 2085-2088) were primed with Pam3CSK, and IL-1β secretionwas measured in response to oxPAPC. The absence of a functional TLR4 didnot alter the capacity of oxPAPC to induce IL-1β release (FIG. 52E).These data further confirmed that there was no requirement for oxPAPC tosignal through TLR4, and that oxPAPC activated DCs upon contact with TLRligands that were indicative of either bacterial or viral infection.Caspase-11 could therefore be classified as a receptor that controlsimmune responses to multiple types of pathogens, not just togram-negative bacteria.

To further examine this possibility in an infectious setting, wild type(WT) or caspase-11 KO mice were infected with herpes simplex virus type1 (HSV-1). HSV-1 was considered to be a good pathogen to examine,because HSV-1 infections activate NLPR3 inflammasomes in ocular modelsof infection (Gimenez, F. et al., (2015). Journal of leukocyte biology2015 Oct. 29. pii: jlb.3HI0715-321R), but this virus does not encodeLPS. Whether caspase-11 was involved in HSV-1 infection was previouslyunknown.

It was identified that caspase-11 KO mice were more susceptible to HSV-1than WT mice on day 2 after ocular infections. Indeed, an increasedabundance of infectious virus was detected in eye swabs from caspase-11,as compared to WT mice, at this time point (FIG. 52F). This differencein viral replication on day 2 was consistent with prior workdemonstrating that NLRP3 KO mice yielded higher viral titers at thistime point (Gimenez, F. et al., (2015). Journal of leukocyte biology2015 Oct. 29. pii: jlb.3HI0715-321R). Subsequent time points resulted inthe clearance of virus from the eye of all mice examined, presumablybecause of the natural transition of the virus to the nervous system.These findings indicated that caspase-11 contributed to the protectionof mice from a non-bacterial pathogen. Without wishing to be bound bytheory, the simplest model to explain these findings was that oxPAPCproduction at the site of infection (the eye) contributed to caspase-11activation and subsequent restriction of viral replication. Developmentof reagents that specifically ablate oxPAPC activities in vivo werecontemplated as necessary for direct testing of this model.

Example 6: Caspase-11 was Identified as a Receptor for oxPAPC

oxPAPC has been shown to have the ability to activatecaspase-11-dependent responses, which indicates an interaction betweenthese molecules. As previously described (Shi, J., et al., (2014b)Nature), endogenous caspase-11 can be isolated from cell lysates throughinteractions with biotinylated-LPS (FIG. 46D). Interestingly,biotin-oxPAPC also formed a complex with endogenous caspase-11 (FIG.46D). In contrast, neither lipid captured endogenous caspase-3 (FIG.46D). To determine whether oxPAPC bound directly to caspase-11, in vitroprotein-lipid interaction studies were performed. As shown in FIG. 46E,oxPAPC displayed a dose-dependent resonance signal with immobilizedcatalytically inactive caspase-11(C254A) on surface plasmon resonance(SPR). In contrast, DMPC, which did not promote IL-1β release from DCs(FIG. 46E), did not bind detectably to caspase-11, and oxPAPC displayedno binding to IgG on the SPR (FIG. 46E). The dissociation constants (Kd)between caspase-11 and oxPAPC were calculated at 1.3×10⁻⁶ M. These SPRdata indicated that caspase-11 formed a complex with a self-encodedlipid (oxPAPC), in addition to LPS, and promoted IL-1β release inresponse to both.

Example 7: LPS and oxPAPC Interacted with Caspase-11 Via DistinctDomains and Induced Different Modes of Activation

Because the same residues within CD14 were required to bind LPS andoxPAPC, whether the LPS-binding CARD was required for interactions withoxPAPC was examined. As expected (Shi et al., 2014b), mutation ofspecific lysine residues within the caspase-11 CARD preventedinteractions with LPS, as assessed by the ability of biotin-LPS tocapture caspase-11 proteins that were produced in 293T cells (FIG. 52G).Interestingly, these lysine residues did not prevent interactions withbiotin-oxPAPC (FIG. 52G). Moreover, a mutant caspase-11 that lacked itsentire CARD, and only contained its C-terminal catalytic domain,retained the ability to form a complex with biotin-oxPAPC (FIG. 52G).SPR analysis verified these results, as the Kd for interactions betweenoxPAPC and the catalytic domain of caspase-11 (noted as 4N59) was nearlyidentical to that calculated for interactions with full lengthcaspase-11 (FIG. 46E). LPS, as expected, had no ability to bind thecatalytic domain of caspase-11. These data therefore established thatunlike CD14, distinct domains within caspase-11 form contacts with LPSand oxPAPC.

In addition to forming a complex with caspase-11, oxPAPC induced theoligomerization of this protein, as indicated by gel filtrationchromatography. As depicted in FIG. 46F, the elution of caspase-11monomers occurred at 15.03 mL, whereas caspase-11 exposed to oxPAPCeluted at earlier volumes, which indicated an increase in the size ofthe protein complex. Dimers of caspase-11 were estimated to elute at13.82 mL, and higher order oligomers were estimated to elute earlier.The ability of oxPAPC to induce the early elution of caspase-11therefore indicated its ability to induce dimerization and/oroligomerization of this protein. The degree of oxPAPC-induced caspase-11oligomerization was less than what was reported for the same activitiesin response to LPS (Shi, J., et al., (2014b) Nature).

LPS-induced oligomerization has previously been shown to promote theintrinsic protease activity of caspase-11 (Shi, J., et al., (2014b)Nature). Because LPS and oxPAPC oligomerize caspase-11 throughinteractions with different domains, caspase-11 enzymatic activity inresponse to each of these lipids was examined. As shown in FIG. 52H, itwas identified that the low intrinsic enzymatic activity of caspase-11monomers was increased upon exposure to LPS or oxPAPC, with LPS as amuch more robust activator.

Without wishing to be bound by theory, there were two possibleexplanations for the minimal ability of oxPAPC to activate caspase-11enzymatic activity. The first possibility was that because oxPAPCpossessed weaker affinity for, and a weaker ability to oligomerize,caspase-11 than LPS, minimal caspase-11 activation occurred. In thisregard, oxPAPC would have simply represented a weak version of LPS.However, the differential mechanisms by which oxPAPC and LPS engagecaspase-11 indicated that these lipids engaged caspase-11 infundamentally different manners, and that the interactions betweenoxPAPC and the catalytic domain were likely designed to obstruct (ratherthan activate) enzymatic activity. Intrinsic enzymatic activity ofpre-existing caspase-11 oligomers was high (FIG. 52H), and this activityincreased further upon exposure to LPS, but notably, this activity wasdiminished upon exposure to oxPAPC (FIG. 52H). Moreover, the ability ofLPS to enhance the enzymatic activity of caspase-11 was blocked byoxPAPC, in a dose-dependent manner (FIG. 52I). These data supported theidea that two distinct biochemical interactions occurred betweencaspase-11 and inflammatory lipids. LPS induced strong oligomerizationand enzymatic activity upon binding the CARD of caspase-11. In contrast,oxPAPC bound the catalytic domain of caspase-11, which promotedoligomerization, but limited enzymatic activity. Despite these twodistinct modes of interaction, both LPS and oxPAPC assembledinflammasomes in DCs, and both promoted IL-1β release.

These findings raised the question of whether the catalytic activity ofcaspase-11 was needed for oxPAPC to induce IL-1β release. To addressthis question, caspase-11-deficient DCs were reconstituted with WT orcatalytic mutant (C254A) caspase-11 expression vectors or empty vector(as control). Cells expressing WT caspase-11 regained the ability torelease IL-1β in response to either LPS or oxPAPC, whereas cellsexpressing mutant caspase-11 did not release IL-1β in response to LPS(FIG. 46G). Interestingly, mutant reconstituted DCs produced as muchIL-1β in response to oxPAPC as did cells expressing WT caspase-11 (FIG.46G). TNFα release was used as a control (data not shown). These dataestablished the differential requirement of caspase-11 activity forIL-1β release in response to LPS versus oxPAPC.

The capacity of mutant and WT caspase-11 to induce pyroptosis, anotherfunction regulated by the non-canonical inflammasomes was also assessed.The enzymatic activity of caspase-11 was necessary for transfected LPSto induce pyroptosis (FIG. 46G), which further confirmed the correctreconstitution of the cells. Surprisingly, no cell death in response tooxPAPC (FIG. 46G) was measured. These data therefore supported two modesof caspase-11 mediated IL-1β release, with the catalytic activity onlybeing necessary for responses to LPS.

Example 8: CD14 Captured and Delivered oxPAPC to Caspase-11 and PromotedIL-1R Release

The above studies indicated that oxPAPC possessed the following twoactivities: 1) it promoted CD14 endocytosis and 2) it promotedcaspase-11 dependent non-canonical inflammasome activation. To determinethe relationship between these activities, the requirement of CD14 foroxPAPC-induced IL-1β release was examined. Cells were primed with LPSfor 3 hours, which was sufficient to permit the re-population of theplasma membrane with newly synthesized CD14 (Tan, Y. et al., (2015).Immunity 43, 909-922), and stimulated with either ATP or oxPAPC.Interestingly, CD14 was required for oxPAPC-induced IL-1β release, asCD14K0 DCs released no IL-1β in response to LPS/oxPAPC or Pam3CSK/oxPAPCtreatments (FIG. 47A). IL-18 secretion, another cytokine released viathe actions of inflammasomes, followed a similar pattern (FIG. 53A).Similar results were obtained when stimulated DCs that were firstisolated from the spleens of WT, CD14 or caspase-11 KO mice wereexamined. These cells exhibited a CD14 and caspase-11 dependent releaseof IL-1β in response to LPS/oxPAPC, whereas TNFα secretion and theupregulation of MHC-II and costimulatory molecules were unaffected byCD14 or caspase-11 deficiencies (FIGS. 47B and 53B). All responses ofsplenic DCs to LPS/ATP treatment were also unaffected by CD14 orcaspase-11 deficiencies (FIG. 47B).

Without wishing to be bound by theory, two lines of evidence suggestedthat the requirement of CD14 for IL-1β release was not due to a defectin the priming phase (i.e. TLR signaling). First, the dose of LPS used(1 μg/ml) bypassed the requirement of CD14 for TLR4-induced cytokineexpression, as assessed by analysis of IL-1β transcripts and secretionof TNFα (FIGS. 47A-47C). Second, Pam3CSK-primed DCs also required CD14for oxPAPC-induced IL-1β release, even though Pam3CSK primes cells viaTLR2 and not CD14 (FIGS. 47A and 47C).

In other experimental settings, type I IFNs promoted caspase-11expression and/or activation (Broz, P. et al., (2012). Nature 490,288-291; Case, C. L. et al., (2013). Proc Natl Acad Sci USA 110,1851-1856; Rathinam, V. A. et al., (2012) Cell 150, 606-619). Since CD14promoted IFN expression in response to LPS treatment, as observed by itsrequirement for viperin expression (FIG. 47C), the role of type I IFNswas examined. Pam3CSK/oxPAPC treated DCs secreted IL-1β (FIG. 47A)without inducing the expression of functional type I IFNs, as indicatedby a lack of viperin expression (FIG. 47C). These data indicated thattype I IFNs were not required to regulate IL-1β secretion in response tooxPAPC stimulation. In addition, the defect in IL-1β secretion in CD14KO cells could not be rescued by exposure to recombinant IFNβ (FIG.47D). IFN expression was therefore neither necessary nor sufficient foroxPAPC to activate caspase-11 dependent inflammasomes in DCs.Furthermore, caspase-1, caspase-11, NLRP3 and ASC were all expressed atcomparable levels in stimulated and unstimulated WT and CD14 KO DCs(FIG. 53C), which suggested that CD14 was not required for theexpression of inflammasome regulators. Collectively, these dataindicated that the requirement of CD14 for oxPAPC mediated IL-1β releasewas not due to a requirement for cell priming. CD14 could thereforeexert a direct role in promoting the inflammasome-mediated IL-1βrelease.

To determine the means by which CD14 promoted inflammasome activation,the endocytosis-promoting activities of this LPS receptor wereevaluated. CD14 promoted oxPAPC endocytosis, and CD14 transported oxPAPCinto the cell to promote IL-1β release. This bypassed the requirement ofCD14 by delivering oxPAPC into the cell via alternative means. Thetransfection reagent DOTAP has previously proven to be a useful tool todeliver inflammatory stimuli directly to endosomes and the cytosol(Honda, K., et al., (2005) Nature 434, 1035-1040). WT and CD14 KO DCwere therefore primed with Pam3CSK and then exposed to DOTAP, in complexwith either LPS or oxPAPC. Consistent with past results, LPS was unableto induce IL-1β release when administered to the extracellular media,but DOTAP-mediated delivery of LPS promoted IL-1β release from WT andCD14 KO DC (FIG. 47E). Interestingly, oxPAPC treatments yielded similarresults. Whereas extracellular oxPAPC did not elicit IL-1β release fromprimed CD14 KO DCs, oxPAPC in complex with DOTAP elicited IL-1β releasefrom CD14 KO cells, in a caspase-dependent manner (FIG. 47E). These dataindicated that alternative delivery mechanisms could bypass therequirement of CD14 for oxPAPC-induced IL-1β release. The primaryfunction of CD14 in inflammasome activation was likely therefore todeliver oxPAPC into the cell.

These data suggested that CD14 functioned to deliver LPS to TLR4 at thecell surface, and to deliver oxPAPC to caspase-11 in the cytosol viasome endosomal intermediate. Since endolysosomes are highly degradativeorganelles, it was possible that the delivery of oxPAPC to endosomeswould also consume this lipid, and limit its inflammatory activities.Consistent with this idea, treatment of DCs with the acidificationinhibitor chloroquine, which blocks endolysosomal activity, promotedslightly more release of IL-1β from DCs (FIG. 53D).

Further, LPS promoted IL-1β release from naïve cells when delivereddirectly to the cytoplasm, whereas DOTAP only permitted oxPAPC totrigger IL-1β release from cells primed with a TLR ligand. Thisdifference in the dependence for priming was likely due to the fact thatLPS possessed the ability to prime the cells via TLR4 and activate IL-1βrelease via caspase-11. In contrast, oxPAPC possessed no ability toprime the cells directly, and therefore depended on a TLR stimulus.These data reinforced the idea that the principle of coincidencedetection operates to govern two types of DC activation states. Thefirst activation state was achieved when DCs encountered PAMPs, andresulted in the release of classic TLR-dependent cytokines viaconventional protein secretion. The second, hyperactive, state wasachieved either when DCs encountered DAMPs in the presence of PAMPs(i.e. coincidence detection), or when virulent bacteria delivered LPS tothe cytosol directly (Aachoui, Y., et al. (2013a). Science 339, 975-978;Casson, C. N., et al. (2013). PLoS Pathog 9, e1003400; Hagar, J. A. etal., (2013) Science 341, 12501253).

Example 9: Unlike Other Inflammasome Activators, oxPAPC Did not KillCells

In addition to promoting IL-β release, inflammasome activation has beentypically associated with the induction of cell death (Aachoui, Y.,(2013b). Current opinion in microbiology 16, 319-326). Without wishingto be bound by theory, cell death via non-canonical inflammasomes isbelieved to depend upon the enzymatic activity of caspase-11, which mustcleave at least two proteins, gasdermin d and pannexin-1 (Kayagaki, N.et al., (2015) Nature 526, 666-671; Shi, J. et al., (2015) Nature 526,660-665; Yang, D. et al., (2015) Immunity 43, 923-932). Since oxPAPC didnot require the catalytic activity of caspase-11 to promote IL-11release, it was contemplated that oxPAPC would not kill cells. To assessthis possibility directly, pyroptosis induction after oxPAPCadministration or LPS transfection in LPS-primed DCs was measured.Pyroptosis is characterized by a rapid loss of plasma membraneintegrity, which should release cytoplasmic proteins (and organelles)from the cell body. LDH release in the supernatant was used to assessmembrane permeabilization of the cell population during pyroptosis.LPS/ATP-treated cells released LDH starting 4 hours after treatment(FIG. 48A); this combination was a well-defined activator ofinflammasome-mediated cell death (Aachoui, Y., (2013b). Current opinionin microbiology 16, 319-326). Cells transfected with LPS, independentlyfrom LPS priming, died at later time points than cells treated with ATP(FIG. 48A). Interestingly, all conditions that activated caspase-11(e.g. LPS transfection or oxPAPC treatment), yielded similar amounts ofIL-11 in the supernatant (FIG. 48B), yet only LPS transfection causedLDH release (FIG. 48A). These data indicated that oxPAPC promoted therelease of IL-11 from living cells.

To corroborate these observations, a single cell assay was developedwhich examined viability of cells that contained assembledinflammasomes, as revealed by the presence of ASC-containing aggregates.It was contemplated that living cells should resist staining with Zombiedye, a stain that labels the cytosol of cells whose plasma membraneshave been disrupted. Cells with intact plasma membranes should alsoretain functional organelles. In contrast, pyroptotic cells should havelost their organelles and should stain strongly for Zombie dye. As shownin FIGS. 48C and 48D, cells treated with LPS/ATP contained ASC specks,and these cells lost mitochondria and stained positive for Zombie dye.In striking contrast, cells treated with LPS/oxPAPC contained ASCspecks, but retained functional mitochondria and displayed minimalZombie dye staining (FIGS. 48C-48D). These collective observationsindicated that oxPAPC possessed no capacity to kill cells, and stronglyindicated that oxPAPC induced IL-1β release from living cells. Moreover,not only did oxPAPC not induce pyroptosis, this lipid counteracted theslow acting death pathways activated by LPS in DCs (Zanoni, I. et al.(2009) Nature 460, 264-268). For this experiment, the health ofindividual cells within the population up to 72 hours after treatmentwas assessed by flow cytometry, using the viability stain 7-AAD, whichdetected genomic DNA within cells whose membranes had been permeabilized(Paterson, A. M. et al., (2011) J Immunol 187, 1097-1105). Usingconcentrations of ATP (1 mM) that induced comparable amounts of IL-1βrelease as those elicited by oxPAPC (FIG. 48B), LPS/ATP treatmentdiminished DC viability soon after treatment (FIG. 54A). Remarkably,whereas LPS treatment alone decreased cell viability at extended timepoints (FIGS. 48E-48F), LPS/oxPAPC treatment actually increasedviability of the cell population (FIG. 48F). These data indicated thatoxPAPC treatment interfered with LPS-induced DC apoptosis to promoteviability.

Example 10: oxPAPC was a Potent Adjuvant Supplement that Promoted T-CellMediated Adaptive Immunity

While caspase-11 contributed to the control of an acute viral infection(FIG. 52F), the dual abilities of oxPAPC to promote DC survival andIL-1β release suggested that oxPAPC might also promote DC-mediatedadaptive immune responses. Indeed, the product of caspase-11 activation,IL-1β, has been characterized as possessing several activities thatpromote T-cell activation (Sims, J. E., and Smith, D. E. (2010) Nat RevImmunol 10, 89-102), including rendering these cells resistant toregulatory T cell suppression (Schenten, D. et al. (2014). Immunity 40,78-90). The oxPAPC/LPS mixtures were examined for their ability todisplay potent adjuvant activity in vivo.

To address this possibility, WT, caspase-11 and caspase-1/-11 dKO micewere injected subcutaneously with LPS, ovalbumin (OVA) and/or oxPAPCthat had been emulsified in incomplete Freund's adjuvant (IFA). Thisexact inoculation route has been used to establish the ability of TLRligands to promote T-cell differentiation (Pasare, C., and Medzhitov, R.(2004) Immunity 21, 733-741; Schnare, M. et al., (2001) Nat Immunol 2,947-950). 40 days after injection, CD4+ T-cells were isolated from thedraining lymph nodes and exposed ex vivo to DCs that were pulsed (ornot) with OVA. T-cell activation was then assessed by measuring theabundance of IL-2, IL-17 and IFNγ by ELISA. Restimulations performedwith DC alone (no OVA) did not elicit IL-2, IL-17 or IFNγ, indicatingthat the cytokines released during restimulations resulted from antigenspecific T-cell responses (FIGS. 48G and 54B).

Interestingly, T-cells isolated from mice immunized with LPS/oxPAPCmixtures yielded substantially higher levels of IFNγ and IL-17 releasethan T-cells isolated from mice immunized with LPS (FIGS. 48G and 54B).The ability of oxPAPC to enhance T-cell activation was lost incaspase-11 or caspase-1/-11 dKO mice (FIGS. 48G and 54B), an observationconsistent with all in vitro data presented herein. Similar results wereobtained measuring T-cell activation 7 days after immunization, i.e.during the effector phase of T cell activation (FIG. 54C). Thus, oxPAPCpossessed the capacity to potentiate LPS-mediated T-cell activation in acaspase-11 dependent manner.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-25. (canceled)
 26. A pharmaceutical composition comprising: (i) aTLR4 agonist, a TLR2 agonist, and/or a TLR9 agonist; (ii) an oxidated1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC)species; and (iii) a cancer immunogen.
 27. The pharmaceuticalcomposition of claim 26, wherein the TLR4 agonist is LPS.
 28. Thepharmaceutical composition of claim 26, wherein the TLR4 agonist ismonophosphoryl lipid A (MPLA).
 29. The pharmaceutical composition ofclaim 26, wherein the TLR2 agonist is Pam3CSK.
 30. The pharmaceuticalcomposition of claim 26, wherein the TLR2 agonist is Pam2CSK.
 31. Thepharmaceutical composition of claim 26, wherein the TLR9 agonist is CpG.32. The pharmaceutical composition of claim 26, wherein the oxPAPCspecies is2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium(HOdiA-PC).
 33. The pharmaceutical composition of claim 26, wherein theoxPAPC species is[(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (KOdiA-PC),
 34. The pharmaceuticalcomposition of claim 26, wherein the oxPAPC species is1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine(HOOA-PC).
 35. The pharmaceutical composition of claim 26, wherein theoxPAPC species is2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium(KOOA-PC).
 36. The pharmaceutical composition of claim 26, wherein theoxPAPC species is (1-palmitoyl-2-(5,6epoxyisoprostanoyl)-sn-glycero-3-phosphocholine) (PEIPC).
 37. Thepharmaceutical composition of claim 26, wherein the cancer is selectedfrom the group consisting of sarcoma, lymphoma, leukemia, carcinoma,melanoma, and astrocytoma.
 38. The pharmaceutical composition of claim37, wherein the carcinoma is selected from carcinoma of the breast,carcinoma of the prostate, ovarian carcinoma, carcinoma of the cervix,colon carcinoma, carcinoma of the lung, and combinations thereof. 39.The pharmaceutical composition of claim 38, wherein the astrocytoma isglioblastoma.